ABCMdb

ABCC7 p.Arg334Cys

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Publications

PMID: 11585852 [PubMed] Smith SS et al: "CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction."

No. Sentence Comment

7 Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. Login to comment
60 Because preliminary experiments suggested that the single-channel conductance of unmodified R334C was much reduced from that seen with wt CFTR, we used symmetric bathing solutions containing Ͼ200 mM Cl for these studies. Login to comment
73 Because the single-channel conductance of R334C CFTR before modification by MTSET was very small, we could not rely upon amplitude histograms for reliable estimation of channel amplitudes at varying membrane potentials. Login to comment
76 In a second set of experiments, MTSET was added to the patch pipette before seal formation and allowed to diffuse to the tip after seal formation, so that modification of R334C CFTR channels by MTSET could be monitored in real time. Login to comment
107 The Function of R334C and K335C CFTR Was Modified by External MTSES or MTSET but the Function of R347C and R352C CFTR Was Not Modified by these Polar Thiol Reagents Fig. 3 summarizes the results of experiments in which MTSES, MTSET, or MTSEA (100 ␮M-10 mM) were added to the solution bathing oocytes expressing wt, R334C, K335C, R347C, or R352C CFTR. Login to comment
111 For both R334C and K335C CFTR, the results of modification by the negatively charged reagent (MTSES) generally agreed with those reported by Cheung and Akabas (1996). Login to comment
112 For R334C CFTR, they reported ~40% decrease in the normalized current (@ -100 mV) and we observed ~70% decrease in the conductance (@Erev). Login to comment
115 Cheung and Akabas (1996) reported that application of 1 mM MTSET to oocytes expressing R334C CFTR reduced the normalized current (@ -100mV) by ~54%, and 2.5 mM MTSEA reduced the normalized current by ~35%. Login to comment
116 In contrast, we found that the application of 100 ␮M or 1 mM MTSET to oocytes expressing R334C CFTR resulted in ~110% increase in the conductance, whereas 100 ␮M or 1 mM MTSEA increased the conductance by ~70%. Login to comment
133 Comparison of the effects of MTSES, MTSET, and MTSEA on the conductance of oocytes expressing R334C, K335C, R347C, or R352C CFTR. Login to comment
153 Consistent with such a view, we found that after removing the reagent from the perfusate, the reaction spontaneously reversed over a period of 30 min, as expected if endogenous reducing agents, such as glutathione and free cysteine, were breaking the disulfide bond. In contrast, exposure of an oocyte expressing R334C CFTR to 1 mM MTSEA for 3 min produced an increase in conductance that remained elevated after washing out the reagent until a reducing agent, 1 mM 2-ME, was added to the perfusate (see Fig. 6 A). Login to comment
159 Covalent modification of R334C CFTR was stable, reproducible, and varied with the electrostatic nature of the moiety covalently attached. Login to comment
166 The Effects of Polar MTS Reagents Applied to R334C and K335C CFTR Were Stable and Readily Reversible with 2-ME, which Is Consistent with the Formation of a Mixed Disulfide Bond The results of an experiment in which an oocyte expressing R334C CFTR was exposed to multiple MTS reagents and 2-ME are shown in Fig. 6 A. Login to comment
177 R334C CFTR did not exhibit time-dependent current relaxations. Login to comment
178 (A) Currents evoked by stepping the potential from -100 to ϩ60 mV for 234 ms from an oocyte expressing R334C CFTR after achieving steady-state activation with stimulatory cocktail and after a brief (3 min) exposure to 1 mM 2-ME. Login to comment
181 (D) I-V plots derived from the current traces shown in A-C: (closed circles) unmodified R334C CFTR; (open circles) MTSET modified R334C CFTR; and (closed triangles) MTSES modified R334C. Login to comment
188 Only R334C exhibited an altered conductance in response to thiol-reactive reagents, which is consistent with the working hypothesis that the observed changes were due to the chemical modification of the cysteine substituted for R334. Login to comment
189 The Modification of R334C and K335C CFTR by MTS Reagents Altered Both the Conductance and the Shape of the Current-Voltage Plot In the course of these experiments, it became apparent that a change in conductance of oocytes expressing either R334C or K335C CFTR induced by exposure to MTSET or MTSES was always accompanied by a concomitant change in the shape of the I-V plot. Login to comment
191 Shown in Fig. 7 are the results of a representative experiment in which currents were elicited by step changes in clamping potential from -100 mV to ϩ60 mV for either the unmodified (A), MTSET modified (B), or MTSES modified R334C CFTR (C), and the resulting I-V plots (D). Login to comment
207 As indicated above, modification by MTSES was very poorly reversible with 1 mM 2-ME at pH 7.5, and as with R334C, current relaxations in response to voltage steps were not detected in either the modified or unmodified state. Login to comment
208 The results of covalent modification were qualitatively similar for K335C and R334C CFTR, but the greater magnitude of the effects for the latter construct led us to focus further studies on modification and substitution at position 334. Login to comment
211 Fig. 9 A shows the activated conductance of oocytes expressing R334C CFTR measured just after modification by MTSET plotted against that measured just before modifi- Figure 8. Login to comment
225 The slope of the line is 2.05 and the intercept is near 0, indicating that over the range of 50-250 ␮S the effect of covalent modification was similar and, at pH 7.4, approximately doubled the conductance due to R334C CFTR. Login to comment
226 Also shown are several determinations made after activation by 200 ␮M IBMX that yields approximately half-maximal activation of R334C CFTR (Wilkinson et al., 1996; Mansoura et al., 1998). Login to comment
235 Covalent Modification of R334C by MTSET Was Detectable as an Increased Single-channel Conductance The results of experiments in which single-channel currents were recorded before and after R334C CFTR was Figure 9. Login to comment
237 (A) Conductance of oocytes expressing R334C CFTR determined just after modification by MTSET plotted versus premodification conductance. Login to comment
244 Dashed lines are mean values of RR for modified and unmodified R334C CFTR. Login to comment
246 The single-channel conductance of R334C CFTR was quite low (‫2.1ف‬ pS, Fig. 10 A) even in the presence of elevated bath Cl concentration (210 mM). Login to comment
252 The inset to the I-V plot (Fig. 10 B) shows the distribution of open probabilities calculated for individual R334C CFTR channels recorded from patches detached either before or after exposure to MTSET. Login to comment
255 As a more rigorous test for possible effects of covalent modification on Po, we monitored the modification of individual R334C CFTR channels using pipettes backfilled with a solution containing 100 ␮M Figure 10. Login to comment
256 Covalent modification of R334C increased single-channel conductance. Login to comment
257 (A) Representative traces from an oocyte expressing R334C CFTR in the detached, inside-out patch configuration, Vm ϭ -80 mV. Login to comment
266 The solid lines are the best fit to the data by linear regression and correspond to a single-channel conductance of 1.2 pS for unmodified R334C CFTR (closed triangles) channels and 3.7 pS after MTSET modification (open triangles). Login to comment
268 Inset shows open probability for R334C CFTR channels at Vm ϭ -100 mV before and after treatment with MTSET (see materials and methods). Login to comment
273 Immediately after detaching the patch into a solution containing PKA and ATP, single channels exhibited the low conductance characteristic of R334C CFTR. Login to comment
279 Covalent modification of a single R334C CFTR channel in real time by including MTSET in the patch pipette. Login to comment
283 (C) Po determined before and after covalent modification for four, single R334C CFTR channels. Login to comment
287 pH titration of oocytes expressing R334C CFTR altered both the conductance and the shape of the I-V relation. Login to comment
288 (A) I-V plot from an oocyte expressing R334C CFTR. Login to comment
293 pH Titration of R334C CFTR Altered Both the Conductance and the Shape of the Current-Voltage Relation The thiol moiety associated with a cysteine residue is expected to bear a time-average, partial negative charge, the magnitude of which will depend on the local pH and the pKa of the sulfhydryl group in situ. Login to comment
296 The conductance and the shape of the I-V relation of oocytes expressing R334C CFTR was highly dependent on the bath pH, exhibiting an apparent pKa of 8.17 Ϯ 0.10 (n ϭ 3; Fig. 12 B). Login to comment
298 If the pH-dependent effects seen with R334C CFTR are due to graded changes in the time-averaged, partial negative charge associated the sulfhydryl group at the 334 locus, then the magnitude of the change induced by chemical modification of this locus should be dependent on bath pH. Login to comment
300 Shown in Fig. 13 are the results of an experiment in which the effects of MTSET and MTSES on the conductance of oocytes expressing R334C CFTR were compared at a bath pH of 6.0 (solid bar) and pH 9.0 (open bar). Login to comment
306 Functional effect of covalent modification of R334C CFTR was dependent on the bath pH. Login to comment
310 Shown in Fig. 14 are the results of modifying R334C CFTR with two "neutral" reagents, NEM and MTSHE. Login to comment
314 Exposure of oocytes expressing R334C CFTR to MTSHE at acidic pH produced an effect reminiscent of MTSES, a conductance decrease, as if negative charge had been added. Login to comment
318 pH Titration of R334H CFTR Altered Conductance and the Shape of the I-V Relation It was of interest to compare the response to changes in bath pH of R334C CFTR with that of R334H CFTR. Login to comment
324 Stepwise acidification of the bath led to stepwise increases in the conductance of oocytes expressing R334C or R334H CFTR and corresponding, stepwise increases in the rectification ratio, whereas similar pH changes resulted in only modest changes in the conductance of oocytes expressing wt CFTR. Login to comment
327 The observed changes seen with wt CFTR were opposite of those seen with oocytes expressing R334C or R334H CFTR, suggesting that the pH induced changes due to protonation of R334C or R334H were, if anything, slightly underestimated. Login to comment
328 The distinct pKa`s of the R334C and R334H variants favor the notion that these pH-dependent effects are due to titration of the side chain at position 334, as opposed to a mutation-induced exposure of a titratable site at another locus (Coulter et al., 1995). Login to comment
330 Modification of R334C CFTR with neutral reagents caused pH-dependent changes in conductance. Login to comment
331 (A) Change in conductance of oocytes expressing R334C CFTR when modified with either NEM or MTSHE at either pH 9.0 or pH 6. Login to comment
344 The Effects of Charge Deposition at Position 334 Were Consistent with the Predictions of Charged-vestibule Models for the Anion Conduction Path The results of covalent modification and pH titration of R334C and R334H CFTR, as well as the functional impact of amino acid substitutions at this site, pointed to an important role for the charge at position 334 in determining the conduction properties of CFTR. Login to comment
349 To investigate the hypothesis that the coordinated changes in conductance and I-V shape seen with covalent modification of R334C CFTR could be attributed to a charge-induced change in the conduction properties of Figure 15. Login to comment
364 (A) RR, determined as described in materials and methods, is plotted as a function of the net charge of the amino acid at position 334 at pH 7.4 where glutamic acid (E) is assigned a value of -1, cysteine (C) is assigned a value of -0.12 based on the apparent pKa of 8.17 for R334C CFTR, histidine (H), alanine (A), and glutamine (Q) are neutral, and lysine (K) and arginine (R) are assigned a value of ϩ1. Login to comment
372 The effects of covalent modification of R334C CFTR could be simulated using charged vestibule models (see appendix). Login to comment
373 The data points shown are the same as in Fig. 7 D containing unmodified R334C CFTR (closed circles), MTSET-modified R334C (open circles), and MTSES-modified R334C (closed triangles). Login to comment
377 (closed circles) Unmodified R334C CFTR, ⌿o ϭ 0. Login to comment
378 (open circles) MTSET-modified R334C CFTR, ⌿o ϭ ϩ 50 mV. Login to comment
379 (closed triangles) MTSES-modified R334C CFTR, ⌿o ϭ -10 mV. Login to comment
381 Symbols as in C for unmodified R334C CFTR (⌿o ϭ 0), MTSET-modified R334C CFTR (⌿o ϭ ϩ50 mV), and MTSES-modified R334C CFTR (⌿o ϭ -10 mV). Login to comment
382 (inset) Plots of percent increase (⌬g/ginitial ϫ 100%) in macroscopic CFTR conductance as function of bath Cl concentration for wt CFTR (open triangles) and unmodified R334C CFTR (closed circles). Login to comment
386 The I-V shape characteristic of unmodified R334C CFTR at pH 7.4 was simulated by adjusting the values of ⌿i and ⌿o, but these values must be viewed as arbitrary for at least two reasons. Login to comment
391 Based on the initial best fit to the data for unmodified R334C CFTR, PCl was fixed at 3.4 ϫ 10-7 cm3/s, and the premodification values of ⌿i and ⌿o were set at ϩ33 mV and -9 mV, respectively. Login to comment
392 When these parameters were used to fit the data for covalently modified R334C CFTR, the effect of MTSET (PCl, ⌿i fixed) was attributed to a 24-mV increase in ⌿o and that of MTSES to a 39 mV reduction of ⌿o (Fig. 17 A). Login to comment
397 Titration of R334C and R334H CFTR by varying bath pH was also well described by a continuum model in which the changes in conductance and I-V shape were largely attributed to changes in ⌿o (unpublished data). Login to comment
399 To determine if saturation of anion flow rate might impact the conductance of R334C CFTR we compared the effects on oocyte conductance of raising the Cl concentration in the external bath. Login to comment
404 In contrast, the maximum increase in conductance in oocytes expressing R334C CFTR (unmodified) was ‫%07ف‬ at 350 mM and consistent with an apparent K1/2 of 147 mM, in accord with the notion that Cl binds in the pore of the R334C CFTR, but with lower affinity than that seen with wt CFTR. Login to comment
408 Initially, values of ⌿o and ⌿i were set to zero, the relative heights (see Fig. 17 legend) of equally spaced barriers and wells (without ionic repulsion) were chosen to reproduce the inward rectifying shape of the I-V curve for unmodified R334C CFTR. Login to comment
411 We used two sets of energy values: one chosen to simulate a "low affinity" channel (K1/2 ϭ 115 mM) like R334C CFTR; and the other chosen to simulate a "high affinity" channel (K1/2 ϭ 38 mM) like wt CFTR. Login to comment
417 Fig. 18 A summarizes the data from experiments in which charge changes were effected in R334C and R334H CFTR by means of chemical modification or pH titration. Login to comment
420 Data points include charge changes brought about by thiol modification of R334C CFTR with positively charged reagents (open triangles), and negatively charged reagents (closed triangles), pH titration of R334C CFTR (closed circles), and pH titration of R334H CFTR (open circles). Login to comment
423 Thiol modification of R334C CFTR with positively charged reagents (open triangles) was treated as adding a single positive charge; thiol modification of R334C with negatively charged reagents (closed triangles) was treated as adding a single negative charge; pH titration of R334H CFTR (open circles) was treated as adding a time-average positive charge determined by the bath pH, assuming a pKa of 5.68; and pH titration of R334C CFTR (closed circles) was treated as adding a time-average negative charge determined by the bath pH assuming a pKa of 8.17. Login to comment
429 The points representing covalent modification of R334C by MTSET or MTSES were corrected for a small (14.5%) charge change predicted due to the protonation state of the cysteine at pH 7.4. Login to comment
443 The evidence for a single site of action of thiol reagents and protons is particularly strong for R334C CFTR as we were able to demonstrate a pH-dependent modulation of functional effects of covalent modification. Login to comment
449 The relatively low single-channel conductance seen with R334C CFTR (even at elevated Cl concentrations) before modification with MTSET and in the patient mutation R3334W (Sheppard et al., 1993) is consistent with the hypothesis that R334 is an important determinant of pore conductance. Login to comment
452 It seems reasonable to conclude that the charge at this site is an important determinant of channel function, and although we speculate that R334 may lie in the pore (as discussed in the next section), it is important to note that covalent addition of a positively charged moiety to R334C CFTR (MTSET) did not rescue the wild-type phenotype. Login to comment
453 The single-channel conductance of 3.7 pS seen after MTSET modification of R334C was less than half of that seen with wt CFTR (8.6 pS) in the same experimental setting. Login to comment
456 One of these, R334W, was studied by Sheppard et al. (1993) who reported that single-channel currents were undetectable in patches detached from HeLa cells in symmetric Cl of ‫061ف‬ mM, which is consistent with the reduced conductance reported here for R334C CFTR. Login to comment
473 If we assume that the outward rectification seen with wt CFTR is due in part to block of Cl efflux by intracellular anions, then the inward rectification seen with R334C CFTR could be attributed, in part, to a reduction in this blocking effect in the mutant channel. Login to comment

PMID: 11585853 [PubMed] Liu X et al: "CFTR: covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane."

No. Sentence Comment

2 Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSETϩ). Login to comment
3 Covalent modification of R334C CFTR with MTSETϩ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Login to comment
5 When oocytes were briefly (20 s) exposed to MTSETϩ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSETϩ. Login to comment
7 The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of ~24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. Login to comment
18 One of these cysteine-substituted constructs (R334C) was readily modified by MTS reagents in the external bath and covalent modification gave rise to the changes in anion conduction that could be easily detected in the macroscopic I-V plots recorded in Xenopus oocytes, permitting us to distinguish modified from unmodified-channels. Login to comment
19 R334C CFTR, in conjunction with membrane impermeant thiol reagent, MTSETϩ (Holmgren et al., 1996), offered an opportunity to test directly the hypothesis that activation of Cl-conductance in oocytes expressing CFTR is accompanied by an increase in channel number in the plasma membrane. Login to comment
20 We found that exposure of oocytes expressing R334C CFTR to MTSETϩ for 20 s before activation resulted in labeling of the entire membrane pool of functional channels, suggesting that channels activated by cAMP are resident in the membrane before activation and that activation of Cl-conductance, therefore, is due largely to an increase in the open probability (Po) of CFTR channels. Login to comment
55 R E S U L T S Modification of R334C CFTR by MTSETϩ Is Stable but Reversible The effects of MTSETϩ and MTSES- modification on the conductance of R334C CFTR are documented in the companion paper (see Smith et al., 2001, in this issue) and were briefly summarized in Fig. 1. Login to comment
56 Expression of R334C CFTR in Xenopus oocytes gives rise to cAMP-activated Cl-conductance characterized by modest inward rectification, which is distinct from that seen with expression of wt CFTR that is characterized by modest outward rectification. Login to comment
57 Brief exposure of oocytes expressing R334C CFTR to MTSETϩ results in an approximate doubling of the conductance and a change in the shape of the I-V plot to one that is linear. Login to comment
59 Recordings from excised patches presented in the companion paper (see Smith et al., 2001, in this issue) also demonstrated that MTSETϩ modification increased the single-channel conductance of R334C CFTR. Login to comment
69 I-V relationship of R334C CFTR is modified by MTSET؉ and MTSES-. Login to comment
75 Modification of R334C CFTR by MTSET؉ was stable for at least 5 h. Login to comment
80 also characteristic of R334C CFTR as described in the accompanying paper (see Smith et al., 2001, in this issue) and Fig. 1 and did not change with time. Login to comment
81 The Entire Membrane Pool of R334C CFTR Activated by cAMP Is Accessible to MTSETϩ before Activation The level of CFTR expression used in these studies was such that, before activation by stimulatory cocktail, the conductance of the oocyte membrane was similar to that seen in an oocyte that was not expressing R334C CFTR. Login to comment
84 Fig. 3 contains plots of the conductance (@Erev) versus time obtained from a group of experiments designed to determine if R334C CFTR channel can be modified by externally applied, impermeant thiol reagents in the active as well as the inactive state. Login to comment
87 The efficacy of MTSETϩ modification of R334C CFTR did not depend on the state of activation of CFTR (Fig. 3 B). Login to comment
91 The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with MTSET؉ before the activation. Login to comment
92 Records of gCl versus time obtained from R334C CFTR expressing oocytes that were obtained from the same frog and assayed on the same day. Login to comment
98 of the oocyte to MTSETϩ and 2-ME induced the same response, suggesting that pre-and postactivation exposure to MTSETϩ labeled the same population of R334C CFTR channels. Login to comment
99 To determine if labeling of the entire pool of R334C CFTR channels could be attributed to nonreacted MTSETϩ that might remain after washing and, thus, be present during channel activation, the channels were Figure 4. Login to comment
100 MTSET؉ labeling did not affect the activation and inactivation process of R334C CFTR. Login to comment
106 The entire membrane pool of R334C CFTR channels that were activated by cAMP was labeled with 20-s exposure to MTSET؉ before the activation. Login to comment
115 To determine if labeling with MTSETϩ altered the process of R334C CFTR activation, we first labeled the channels with 100 ␮M MTSETϩ after activation, then inactivated the labeled channels, and then reactivated them at a later time (Fig. 4). Login to comment
118 Reactivation of R334C CFTR increased gCl to a level similar to that seen after the first modification, and 2-ME decreased gCl by ‫.%05ف‬ The effects were reproduced by the second exposure to MTSETϩ and 2-ME, indicating that MTSETϩ did not interfere with the activation or inactivation of R334C CFTR. Login to comment
124 The shape of the I-V plot obtained at steady-state activation in the presence of MTSES- was characteristic of MTSETϩ- modified R334C CFTR (Fig. 5 C, 3), indicating that prelabeling with MTSETϩ for 20 s prevented modification by MTSES- before and during activation. Login to comment
126 Exposure of the oocyte to the reducing reagent (2-ME) decreased the conductance to about half the stimulated value, and changed the shape of the I-V plot to inward rectification typical of unmodified R334C (Fig. 5 C, 6), indicating that the positively charged, TEA group that was added in the inactive state was readily removed. Login to comment
128 The results indicate that the modification of R334C CFTR by MTSETϩ was complete, regardless of whether exposure to the reagent took place in the active or inactive state, confirming that the entire pool of CFTR channels was accessible to MTSETϩ before activation. Login to comment
129 The Time Course of Addition of R334C CFTR Channels to the Plasma Membrane after cRNA Injection After cRNA injection, new CFTR channels must be synthesized and inserted in the oocyte plasma membrane. Login to comment
131 Time-dependent increase in the conductance of R334C CFTR in the first 5 d after cRNA injection. Login to comment
138 Fig. 6 is a summary of the conductance of R334C CFTR in its inactive and active state during the first 5 d after cRNA injection. Login to comment
171 The oocytes used in this group of experiments were obtained from the same frog and were injected with R334C CFTR RNA at the same time. Login to comment
174 D I S C U S S I O N In CFTR-expressing Oocytes cAMP Increases Cl-Conductance by Increasing the Open Probability of Channels The results presented here are consistent with the notion that the entire, activatable pool of R334C CFTR in the Xenopus oocyte is accessible to MTSETϩ during a 20-s exposure to a perfusion solution containing the reagent. Login to comment
187 MTSETϩ Is Impermeant The studies of Holmgren et al. (1996) and Yang et al. (1996) suggested that MTSETϩ is not likely to cross the plasma membrane and label R334C CFTR protein that might exist in subplasma membrane vesicles. Login to comment
209 If endocytosis of R334C CFTR were proceeding in Xenopus oocytes at the highest rate reported for mammalian cells (‫/%05ف‬min), then the maintenance of the steady-state, activated CFTR conductance commonly observed in oocytes would require an equally high exocytotic delivery rate to maintain the channel population. Login to comment
211 This sort of behavior was never observed, suggesting that the rate of turnover of R334C CFTR is relatively slow in Xenopus oocytes. Login to comment
218 It must also be emphasized that all of the results reported here were obtained using a CFTR mutant (R334C), so that it is possible this mutation suppresses stimulation-dependent trafficking mechanism that is more prominent in the wild-type and M2-901 CFTR (see Mechanism of CFTR Activation). Login to comment
219 However, like other membrane proteins that undergo clathrin-mediated endocytosis, the internalization signal contained in the cytoplasmic tail of the wt CFTR (which was retained in R334C CFTR) was found to be sufficient for promoting endocytosis (Prince et al., 1999). Login to comment
220 It also seems unlikely that labeling of R334C CFTR with thiol reagents would cause the apparent low rate of recycling, given that biotinylated CFTR is efficiently endocytosed in mammalian cells (Prince et al., 1994; Lukacs et al., 1997). Login to comment
221 Blocking the Insertion of Channels Did Not Affect Activation After labeling with MTSETϩ, the appearance of unlabeled channels on the plasma membrane was observed during the first few days after the injection of R334C cRNA when the insertion of new channels via the biosynthetic pathway was expected. Login to comment

PMID: 12411425 [PubMed] Gong X et al: "Mechanism of lonidamine inhibition of the CFTR chloride channel."

No. Sentence Comment

7 5 Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) signi®cantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. Login to comment
116 As shown in Figure 7a, 55 mM lonidamine inhibited currents carried by R334C, K335A, F337S, T338A and S341A-CFTR. Login to comment
117 However, R334C, F337S and S341A were only weakly inhibited by this concentration relative to wild-type CFTR (see Figure 1). Login to comment
118 The eect of these mutations on block by lonidamine is more clearly seen in the dose-response curves shown in Figure 7b. Fits of these mean data by equation 1 suggests a Kd (at 7100 mV) of 58.5 mM for wild-type, 65.6 mM for K335A, 90.0 mM for T338A, 186 mM for F337S, 206 mM for S341A, and 338 mM for R334C. Login to comment
119 Similar analyses at other potentials showed a similar increase in Kd in R334C, F337S, S341A and (to a far lesser extent) T338A (Figure 7c). Login to comment
120 Fitting data from individual patches with equation 2 gave similar and, except in the case of K335A, signi®cant changes in Kd(-100): wild-type 60.6+5.2 mM (n=5), K335A 63.1+7.4 mM (n=5) (P40.05), T338A 93.4+4.1 mM (n=5) (P50.002), F337S 166+18 mM (n=5) (P50.0005), S341A 169+25 mM (n=5) (P50.005), R334C 260+19 mM (n=4) (P50.00001). Login to comment
121 These same ®ts also revealed changes in the voltage dependence of block, as judged by changes in d, although this was only statistically signi®cant in the case of R334C: wild-type 0.426+0.033 (n=5), K335A 0.484+0.024 (n=5) (P40.05), T338A 0.410+0.045 (n=5) (P40.05), F337S 0.365+0.015 (n=5) (P40.05), S341A 0.285+0.061 (n=5) (P40.05), R334C 0.233+0.066 (n=4) (P50.05). Login to comment
143 (a) Example I-V relationships for R334C, K335A, F337S, T338A and S341A-CFTR, before (solid lines) and following (dotted lines) addition of 55 mM lonidamine to the intracellular solution. Login to comment
145 (b) Concentration dependence of block at 7100 mV for wild-type, R334C, K335A, F337S, T338A and S341A. Login to comment
147 Each has been ®tted by equation 1, giving Kds of 58.5 mM (wild-type), 65.6 mM (K335A), 90.0 mM (T338A), 186 mM (F337S), 206 mM (S341A) and 338 mM (R334C). Login to comment
154 Lonidamine block was weakened in the TM6 mutants R334C, F337S and S341A (Figure 7), suggesting that these residues may normally contribute to lonidamine binding within the pore. Login to comment
157 The strong eects of the R334C mutant (Figure 7) are somewhat surprising, given that this residue is purportedly in the external pore vestibule. Login to comment

PMID: 12679372 [PubMed] Gong X et al: "Molecular determinants and role of an anion binding site in the external mouth of the CFTR chloride channel pore."

No. Sentence Comment

43 In contrast, an additional mutation near the external end of TM6, R334C, not only caused a more dramatic weakening of Au(CN)2 _ block than previously studied mutants (Fig. 1), but also abolished or even reversed the Cl_ dependence of both apparent affinity (as judged by the Kd(0); Fig. 2A) and voltage dependence (as judgedbythefractionalelectricaldistance,d;Fig.2B). Login to comment
46 Whereas in wild-type, Kd(0) increases dramatically as the extracellular anion is made more lyotropic (gluconate å Cl_ å SCN_ ), this trend is apparently abolished in R334C (Fig. 3C). Login to comment
49 CharacterizationofR334asananionbindingsite The effects of the mutation R334C outlined above suggest that the positively charged arginine at this position normally contributes both to a lyotropic binding site and to a site which is crucial for electrostatic interactions between extracellular permeant ions and intracellular blocking ions. Login to comment
51 Previously we reported that the mutant R334A could not be expressed in BHK cells (Gong et al. 2002a); in the course of the present study, we confirmed this previous finding, but did find that, in addition to R334C, five other mutants could be studied (Fig. 4). Login to comment
53 Block of wild-type, R334C-, R334E-, R334H-, R334K-, R334L- and R334Q-CFTR by 100 mM and 1 mM intracellular Au(CN)2 _ are compared in Fig. 4B. Login to comment
54 Block was affected in all mutants, depending on the ionic conditions used, but was particularly weakened in R334C, R334E and R334K (Fig. 5A-C). Login to comment
61 In contrast, in R334C extracellular Cl_ significantly weakens block and reduces its voltage dependence. Login to comment
65 Au(CN)2 _ block of R334C is relatively independent of the extracellular anion A, example R334C-CFTR I-V relationships recorded before (control) and after (+Au(CN)2) addition of 1 mM Au(CN)2 _ to the intracellular solution, with 150 mM NaCl, sodium gluconate or NaSCN present in the extracellular solution. Login to comment
67 C, Kd(0) is strongly affected by the extracellular anion in wild-type (black bars) but not in R334C (grey bars). Login to comment
68 D, the effect of changing the extracellular anion (from Cl_ to gluconate or from Cl_ to SCN_ ) on the strength of Au(CN)2 _ block, as quantified by the ratio of Kd(0) values estimated under different ionic conditions as described in Methods, is significantly reduced in R334C (grey bars) relative to wild-type (black bars) (**P < 0.001). Login to comment
111 Each of the mutations R334C, R334E and X. Gong and P. Linsdell394 J Physiol549.2 Figure 8. Login to comment
122 Previously we showed that the R334C mutation significantly weakened block by intracellular lonidamine (Gong et al. 2002b), consistent with the idea that permeant and blocking ions may share common binding sites within the pore. Login to comment
141 With either Cl_ or gluconate in the extracellular solution, Au(CN)2 _ block was most dramatically weakened in the mutants R334C, R334E and R334K, which involve replacement of the positively charged arginine side chain with one neutral side chain (cysteine), one negatively charged side chain (glutamate) and one positively charged side chain Anion binding site in the CFTR pore outer mouthJ Physiol 549.2 395 (lysine). Login to comment

PMID: 12745925 [PubMed] Gupta J et al: "Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore."

No. Sentence Comment

33 However, in contrast to the linear macroscopic I Á/V relationship observed for wild-type CFTR under symmetrical ionic conditions, most mutants showed some degree of inward rectification (Figure 1B); this rectification was particularly strong for R334C. Login to comment
41 Example leak-subtracted I Á/V relationships obtained with different intracellular anions are shown for wild-type, R334C, F337A, T338A, T339V and S341A in Figure 2. Login to comment
44 Of eight mutants studied, only T339V was without any significant effect on anion permeability (Table 1), and five mutations (R334C, K335A, F337A, T338A, I340A) led to changes in the permeability sequence among halides (Figure 2 and Table 2). Login to comment
59 Wild type R334C K335A I336A F337A T338A T339V I340A S341A Cl 1.009/0.00 (6) 1.009/0.01 (6) 1.009/0.05 (5) 1.009/0.01 (5) 1.009/0.02 (6) 1.009/0.02 (8) 1.009/0.03 (6) 1.009/0.02 (5) 1.009/0.01 (6) Br 1.479/0.06 (6) 0.969/0.00 (5)** 1.529/0.03 (5) 1.359/0.05 (5) 0.669/0.03 (6)** 2.209/0.05 (5)** 1.829/0.24 (5) 1.409/0.09 (6) 2.459/0.20 (5)** I 0.819/0.04 (6) 0.729/0.05 (3) 1.579/0.06 (4)** 0.589/0.02 (4)* 0.389/0.15 (3)* 2.799/0.26 (7)** 0.769/0.02 (6) 1.249/0.07 (6)** 0.739/0.06 (6) F 0.119/0.01 (6) 0.099/0.01 (3) 0.139/0.02 (3) 0.079/0.01 (5) 0.409/0.02 (4)** 0.139/0.01 (6) 0.079/0.00 (5) 0.069/0.01 (5) 0.059/0.01 (6)* SCN 4.759/0.30 (6) 2.769/0.38 (6)** 3.989/0.16 (5) 3.709/0.11 (5)* 1.269/0.12 (5)** 7.509/0.29 (6)** 4.829/0.40 (5) 4.189/0.14 (7)* 10.09/1.8 (6)* Relative permeabilities for different anions present in the intracellular solution under bi-ionic conditions were calculated from macroscopic current reversal potentials according to Eq. (1) (see Experimental procedures). Login to comment
65 Wild-type R334C K335A I336A F337A T338A T339V I340A S341A Cl (G(50/G'50) 1.039/0.09 (6) 4.509/0.60 (6)** 1.399/0.09 (5)** 1.519/0.14 (5)* 1.189/0.22 (6) 1.779/0.25 (8)* 1.199/0.06 (7)* 1.419/0.11 (5)* 1.809/0.18 (5)** Cl (GCl/GCl) 1.009/0.08 (6) 1.009/0.13 (6) 1.009/0.07 (5) 1.009/0.09 (5) 1.009/0.22 (6) 1.009/0.14 (8) 1.009/0.06 (7) 1.009/0.09 (5) 1.009/0.10 (5) Br 0.649/0.05 (6) 0.329/0.02 (6)** 0.669/0.05 (5) 1.079/0.10 (5)* 0.359/0.06 (6)** 0.499/0.03 (5) 0.659/0.09 (5) 0.669/0.08 (6) 1.529/0.30 (4)* I 0.299/0.05 (6) 0.749/0.02 (3)* 0.279/0.01 (4) 0.109/0.02 (4)* 0.349/0.08 (3) 0.389/0.03 (5) 0.309/0.05 (7) 0.279/0.03 (6) 1.049/0.16 (7)** F 0.379/0.04 (6) 0.329/0.04 (3) 0.349/0.03 (3) 0.709/0.10 (4)* 0.129/0.02 (3)* 0.239/0.02 (6)* 0.509/0.10 (4) 0.309/0.02 (5) 0.519/0.07 (6) SCN 0.389/0.02 (6) 0.339/0.03 (6) 0.669/0.10 (5)* 0.279/0.02 (6)* 0.399/0.04 (5) 0.269/0.02 (5)* 0.269/0.02 (4)* 0.359/0.04 (6) 0.839/0.14 (6)* Relative conductances for different anions were calculated from the slope of the macroscopic I Á/V relationship for inward versus outward currents (see Experimental procedures). Login to comment
73 Conversely, the sequence is changed to Eisenman sequence IV in R334C and Eisenman sequence V in F337A, consistent with relative loss of lyotropic anion selectivity in these mutants. Login to comment
77 Smaller but statistically significant decreases in PSCN/PCl were observed in R334C, I336A and I340A. Login to comment
78 Taken together, these anion permeability data suggest a relative loss of lyotropic anion selectivity in F337A and (to a lesser extent) R334C, strengthening of lyotropic selectivity in T338A and S341A, and only minor effects at other positions. Login to comment
86 Halide permeability sequence Eisenman sequence CFTR variants I( !/Br( !/Cl( !/F( I K335A, T338A Br( !/I( !/Cl( !/F( II I340A Br( !/Cl( !/I( !/F( III wild-type, I336A, T339V, S341A Cl( !/Br( !/I( !/F( IV R334C Cl( !/Br( !/F( !/I( V F337A Sequences were derived from the relative permeabilities given in table 1. Login to comment
102 Current rectification was particularly striking in R334C (Figure 1B), consistent with the recently described role of the positive charge at this position in attracting anions to the mouth of the pore [26]. Login to comment
109 Lyotropic anion selectivity is disrupted in F337A and modified in R334C, T338A and S341A. Login to comment
124 In most (six of eight) cases, alanine substitution was employed; however, we have previously found that the mutants R334A [15] and T339A [22] fail to express in BHK cells, and for these residues mutants which gave adequate current expression (R334C, T339V) were studied. Login to comment

PMID: 14581585 [PubMed] Cai Z et al: "Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl- channel."

No. Sentence Comment

397 Third, comparison of the data of Smith et al. (2001) and Gong and Linsdell (2003) suggests that the strong inward rectification of the CFTR variant R334C is not accounted for by voltage-dependent changes in i. Based on these data, we argue that voltage-dependent changes in CFTR channel gating contribute to the inward rectification of macroscopic CFTR Cl- currents. Login to comment

PMID: 14598388 [PubMed] Liu X et al: "CFTR: what's it like inside the pore?"

No. Sentence Comment

93 No functional impact was observed in wild type CFTR upon exposure to these reagents, but covalent charge changes at position 334 (R334C CFTR) produced charge-dependent effects on macroscopic conductance and the shape of the I-V plot. Login to comment
96 Summarized in Figure 4 are charge-induced changes in R334C or R334H CFTR conductance that result from alteration of external pH or exposure of oocytes expressing R334C CFTR to charged methanethiosulfonate (MTS) reagents. Login to comment
98 Acidification of the bath solution of oocytes expressing R334C or R334H CFTR, or modification of R334C CFTR by a positively charged MTS Fig. 4. Login to comment
103 (B) pH titration of R334C. Login to comment
108 On the other hand, alkalinization of the bath solution of oocytes expressing R334C or R334H CFTR or modification of R334C CFTR by a negatively charged MTS reagent, MTSES (sodium [2-sulfonatoethyl]methanethiosulfonate), decreased the whole cell conductance and enhanced the inward rectification of the shape of the I-V plots. Login to comment
110 MTSET modification of R334C CFTR conductance caused an appropriate doubling in single channel conductance and had no marked effect on the open probability of the channel (Smith et al., 2001). Login to comment
118 Changing the charge at position 334 either by modification of R334C/T338H CFTR with polar thiol reactive reagents or by amino acid substitution (R334A/T338C) shifts the titration curve in a direction that was predicted on the basis of a nearby positive charge being able to stabilize a titratable group (Liu et al., 2001). Login to comment

PMID: 14610019 [PubMed] Gong X et al: "Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore."

No. Sentence Comment

98 Block of R334C and S341A appeared somewhat weaker than for wild-type CFTR, whereas K335A and T338A showed a similar degree of block as wild-type (Fig. 5, A-C). Login to comment
145 (A) Example macroscopic currents carried by the CFTR mutants R334C, K335A, F337A, T338A, and S341A before (Control) and after addition of 300 ␮M Pt(NO2)4 2to the intracellular solution. Login to comment
147 Each plot has been fitted by Eq. 2; this provides a good fit of R334C (Kd(0) ϭ 2080 ␮M, z␦ ϭ -0.174), K335A (Kd(0) ϭ 418 ␮M, z␦ ϭ -0.317), T338A (Kd(0) ϭ 626 ␮M, z␦ ϭ -0.351) and S341A (Kd(0) ϭ 1362 ␮M, z␦ ϭ -0.249), but a poor fit of F337A. Login to comment

PMID: 15229107 [PubMed] Chen Y et al: "Mechanism of activation of Xenopus CFTR by stimulation of PKC."

No. Sentence Comment

33 XCFTR R334C was generated by substitution of Arg334 with Cys using the QuickChange multisite-directed mutagenesis kit (Stratagene, La Jolla, CA). Login to comment
126 Oocytes expressing XCFTR-R334C and wild-type XCFTR had similar responses to PMA. Login to comment
146 To test whether PMA stimulation causes insertion of new XCFTR channels, oocytes expressing the XCFTR-R334C mutant were first exposed to MTSET for 10-20 s, then the MTSET was thoroughly washed out and the oocytes were stimulated with PMA. Login to comment
163 Effects of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) on Gm of oocytes expressing XCFTR-R334C. Login to comment

PMID: 15282191 [PubMed] Chen Y et al: "Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2."

No. Sentence Comment

56 The mutations to generate R334C XCFTR and XCFTR-hCFTR chimeras (see Fig. 1) were introduced with the QuickChange Multi Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), with all subcloning done through the plasmid pCR-4Blunt- TOPO with PCR-amplified CFTR DNA fragments. Login to comment
58 The mutagenic primer to generate the R334C mutant was 5Ј-GGCATT- TCACTCTGTAAGATCTTTACTACCATTTCATTTAGC-3Ј. Login to comment
143 In control experiments, the increase in Gm elicited by the thiol reagent MTSETϩ (1 mM) was 98 Ϯ 5% (n ϭ 6) in activated XCFTR-R334C (Fig. 4, A and B). Login to comment
146 To test for channel insertion, oocytes expressing XCFTR-R334C were exposed to MTSETϩ for 10-20 s during the first PKA stimulation and then the agent was washed out from the bath. Login to comment
149 As illustrated in Fig. 4C, when oocytes expressing XCFTR-R334C were stimulated by PKA subsequent to PKA and PKC stimulation, there was no appreciable change in conductance with the second exposure to MTSETϩ . Login to comment
192 [2-(Trimethylammonium)ethyl]methanethiosulfonate bromide (MTSETϩ ) modifies the Gm of oocytes expressing XCFTR-R334C. Login to comment

PMID: 15504728 [PubMed] Zhang ZR et al: "Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore."

No. Sentence Comment

3 Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. Login to comment
5 The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. Login to comment
20 EXPERIMENTAL PROCEDURES Preparation of Oocytes and cRNA-For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers (14). Login to comment
21 R334C was prepared from a construct carrying the full coding region of CFTR in the pBluescript vector. Login to comment
43 Since preliminary experiments showed that the full single channel conductance of unmodified R334C-CFTR was very low (1.5 pS) compared with that of WT-CFTR (14, 15), most single-channel experiments in this study used asymmetrical [Cl- ] in order to increase the single channel amplitude at VM ϭ -100 mV. Login to comment
64 Analysis of Single Channel and Macropatch Experiments-The Fetchan and pSTAT programs of pClamp 8.0 were used to calculate open probability (Po) and to make all-points amplitude histograms for R334C-CFTR channels before and after modification. Login to comment
71 As described below, R334C channels exhibit multiple conductance levels, with s1 representing subconductance level 1, s2 being subconductance level 2, and f being full conductance level, as well as the closed level (c). Login to comment
75 It is noteworthy that because the single channel amplitude of R334C is very small, we transferred the all-points histogram data for R334C-CFTR channels to PeakFit version 4.11 (SYSTAT Software Inc., Chicago, IL) to verify the result of fits in pClamp 8.0. Login to comment
93 Fig. 1 also contains records illustrating the subconductance behavior of two mutant CFTRs: R334C and T338A. Login to comment
97 This result suggests that neither the R334C nor T338A mutation, although they involve residues reputed to lie within the CFTR pore (14, 18), greatly altered the relative magnitude of the subconductance states. Login to comment
99 In a longer record of single-channel currents from oocytes expressing R334C-CFTR (Fig. 2, A and B), the most prominent state is discernible as a conductance of ϳ1.2 pS that was reported previously (14), although upon closer examination of the fine structure of the open bursts, it is found that within nearly every burst there are transitions to three conductance states: one lower in conductance than that of the most frequent state and one higher in conductance. Login to comment
101 Deposition of a Positive Charge at 334 Amplifies All Conductance States Proportionately-We reasoned that if the three conductance states reflect different behaviors of a shared portion of the conduction path, which includes the amino acid at position 334 in TM6, it would be possible to use the properties of the R334C mutant to investigate the architecture of the functional CFTR pore. Login to comment
102 We showed previously that covalent modification of R334C-CFTR channels with MTSETϩ increased the amplitude of the most prominent single-channel conductance (referred to here as s2) without altering gating (14). Login to comment
107 R334C-CFTR chloride channels were modified in ϳ15 min by MTSETϩ diffusing down the electrode tip, as reflected by an increment of s1, s2, and f conductance levels ϳ2.12.3-fold (Fig. 2, B-F). Login to comment
108 We analyzed eight paired, inside-out single channel records (both pre-and postmodified channels included in the same patch) that contained only one R334C-CFTR channel per patch, as shown in Fig. 2A. Login to comment
111 We maintained the patches that contained modified R334C-CFTR channels for up to 45 min in some experiments and found that following modification by MTSETϩ , the amplitudes of the s1, s2, and f conductance states consistently stayed at the same level, with no further modification observed. Login to comment
115 Hence, the shared impact of covalent modification by MTSETϩ on the amplitude of all conductance states exhibited by R334C-CFTR channels was also consistent with the hypothesis that the three conductance states have at least a portion of the conduction path in common. Login to comment
116 Covalent Modification of R334C-CFTR Did Not Alter Gating-We previously reported that modification of R334C-CFTR by MTSETϩ did not alter gating as defined by comparing open probability in patches from treated and untreated oocytes as well as that of channels monitored during the process of modification (14). Login to comment
117 Analysis of the subconductance behavior of R334C-CFTR provided an opportunity to reexamine the question of possible gating effects by asking whether modification of R334C-CFTR channels by MTSETϩ altered the prevalence or duration of the three conductance states. Login to comment
119 The overall Po of R334C-CFTR channels before and after MTSETϩ modification was 0.24 Ϯ 0.04 and 0.23 Ϯ 0.04, respectively (p ϭ 0.91; see Fig. 3D). Login to comment
120 Furthermore, as shown in Fig. 2G, the fractional abundance of the s1, s2, and f conductance states did not change upon MTSETϩ -induced modification in R334C-CFTR channels. Login to comment
124 A-C, records for WT-, R334C-, and T338A-CFTR, respectively, were generated in excised, inside-out mode with asymmetrical [Cl- ], where the pipette was filled with 40 mM [Cl- ] and bath (cytoplasmic) solution contained 302 mM [Cl- ] in order to potentiate the single channel amplitude. Login to comment
128 Furthermore, the observation in R334C-CFTR that the amplitudes of the s1, s2, and f states increased by an equivalent proportion, and apparently simultaneously, upon modification by MTSETϩ suggests that each of these states reflects the activity of a single pore, or a portion of a shared conduction pathway rather than the activity of two separate pores. Login to comment
130 Modification of R334C-CFTR increases the conductance of each open state. Login to comment
131 A, representative trace from an oocyte expressing R334C-CFTR in the detached, inside-out patch configuration during real time modification; VM ϭ -100 mV, with asymmetrical [Cl- ]. Login to comment
136 B and C, two isolated single bursts representing R334C-CFTR from the same patch before (B) and after (C) modification by MTSETϩ . Login to comment
156 A similar result was obtained using a double mutant, R334C/K1250A, that exhibits a prolonged open state duration (data not shown). Login to comment
163 In Fig. 4, activated R334C-CFTR channels were first exposed to the bath solution containing no MTSETϩ for a time period of ϳ20 s and then perfused by bath solution containing 50 ␮M MTSETϩ . Login to comment
164 R334C-CFTR macroscopic current increased rapidly, reflecting modification by MTSETϩ (14). Login to comment
165 The final, steady-state macroscopic current amplitude of modified R334C-CFTR was increased by 2.3 Ϯ 0.22-fold after prolonged exposure to 50 ␮M MTSETϩ . Login to comment
171 This observation provided an opportunity to test directly the notion that a nearby positive charge would modify the rate of modification of the cysteine at 334 by comparing the rate of modification of R334C-CFTR with the rate of modification of R334C/K335A-CFTR (Fig. 4B). Login to comment
172 The amplitude of macroscopic current was increased 2.97 Ϯ 0.24-fold by 50 ␮M MTSETϩ in R334C/K335A-CFTR. Login to comment
175 Modification of R334C-CFTR does not affect reversal potential and open probability. Login to comment
177 D, Po determined before (filled circles) and after (filled triangles) covalent modification by MTSETϩ for eight paired patches containing single R334C-CFTR channels. Login to comment
180 in the vicinity of R334C. Login to comment
182 As an additional test for the presence of multiple cysteines, we studied the kinetics of modification of R334C-CFTR channels in outside-out macropatches using a two-pulse protocol as follows. Login to comment
189 If there were two cysteines in each one-pore CFTR, then following the first brief exposure to MTSETϩ at a low concentration, the pool of R334C-CFTR channels should comprise a mixed population of unmodified, singly modified, and doubly modified channels (Fig. 5C); longer exposure to MTSETϩ in the first treatment would lead to a greater increase in current, due to modification of more cysteines. Login to comment
190 The change in electrostatic potential due to modification of one cysteine would be expected to alter the rate of modification of the remaining cysteine, as suggested by the difference in response in R334C- and R334C/ K335A-CFTR. Login to comment
194 Outside-out macropatch experiments for mutants R334C- and R334C/K335A-CFTR. Login to comment
197 A, an example of macroscopic current of R334C-CFTR, filtered at 200 Hz. Login to comment
199 The amplitude of macroscopic current was increased by 2.3-fold upon modification by MTSETϩ in this experiment. B, representative macroscopic current of R334C/K335A-CFTR; the red line is the curve fit, with ␶ ϭ 1.1 s in this experiment. Login to comment
201 R334C-CFTR channels have one engineered cysteine per pore. Login to comment
202 A, outside-out macropatch experiment for R334C-CFTR, using a protocol similar to that shown in Fig. 4 but with two exposures to MTSETϩ . Login to comment
206 In experiments such as that shown in Fig. 5A, brief exposure to 5-10 ␮M MTSETϩ should modify a subset of the available cysteines, resulting in one of three conditions: (i) in some R334C-CFTR pores, neither of the cysteines would be modified; (ii) in some pores, only one cysteine would be modified; and (iii) in some pores, both of the two cysteines within a single pore would be modified (stars indicate the cysteines that were modified by MTSETϩ ). Login to comment
213 To determine whether any engineered cysteines remain unmodified in R334C-CFTR channels after prolonged exposure to MTSETϩ , we took advantage of the sensitivity of unmodified cysteines to bath pH. Login to comment
214 R334C-CFTR channels were examined by two-electrode voltage clamp, and channels were activated via the beta2-adrenergic receptor by exposure to isoproterenol. Login to comment
215 As reported previously, the conductances of oocytes expressing unmodified R334C-CFTR channels were sensitive to bath pH, due to titration of the partial negative charge on the unmodified cysteine (14). Login to comment
217 The macroscopic conductance of R334C-CFTR was increased ϳ2.5-fold (n ϭ 3, Fig. 6B) upon MTSETϩ -induced modification at bath pH 7.5, which is consistent with our previous report (14). Login to comment
218 However, after R334C-CFTR channels were covalently modified by 200 ␮M MTSETϩ , the macroscopic conductance was no longer sensitive to pH titration (Fig. 6, B and D). Login to comment
221 The possibility remains, however, that two separate copies of R334C contribute to each functional pore and that MTSETϩ - induced modification of these two targets occurs with identical rates as expected if the two sites are far enough apart in the folded channel polypeptide that the electrostatic charge change that accompanies modification of one site is not sensed at the other site. Login to comment
224 Although our previous experiments showed that the number of R334C-CFTR channels resident at the oocyte plasma mem- FIG. 6. Login to comment
225 R334C-CFTR was not accessible to protons after modification by MTSET؉ . Login to comment
226 A and C, oocytes expressing R334C-CFTR and beta2-adrenergic receptor were first activated by ND96 plus 5 ␮M isoproterenol at pH 7.5 for 6 min. Login to comment
228 On average, the macroscopic conductance of R334C-CFTR increased 22 Ϯ 3% (p ϭ 0.02, n ϭ 3) in pH 6.0 and decreased 50 Ϯ 5% (p ϭ 0.01, n ϭ 3) in pH 9.0. Login to comment
229 B and D, oocytes expressing R334C-CFTR and beta2-adrenergic receptor were first activated by ND96 plus 5 ␮M isoproterenol at pH 7.5 for 6 min and then followed by the same solution containing 200 ␮M MTSETϩ for 4 min; the macroscopic conductance was increased by ϳ2.5-fold upon application of MTSETϩ . Login to comment
230 Modification by MTSETϩ prevented the pH-induced response seen in R334C-CFTR macroscopic conductance. Login to comment
232 To control for potential changes in channel number, we analyzed single-channel recordings containing multiple R334C-CFTR channels in excised mode, while MTSETϩ diffused down to the tip from a back-filled pipette; the example shown in Fig. 7 contained at least three active channels. Login to comment
243 R334C-CFTR channels contain a single population of engineered cysteines. Login to comment
244 A, an individual patch containing at least three R334C-CFTR channels. Login to comment
246 The upper trace includes the first modified R334C-CFTR opening, indicated by the filled arrowhead. Login to comment
247 Through time, the number of unmodified R334C-CFTR openings was reduced (middle trace, indicated by the open arrowheads), and finally all openings exhibited the modified conductance (bottom trace). Login to comment
284 In the present experiments, we compared the subconductance behavior of wild type and mutant CFTRs and investigated the effect on subconductance behavior of covalent charge deposition using R334C-CFTR. Login to comment
291 Neither the impact of covalent labeling nor the kinetics of labeling provided any evidence for the presence of more than a single cysteine in the pore formed by R334C-CFTR. Login to comment
296 First, functional modification of R334C-CFTR by reagents such as MTSETϩ and 2-sulfonatoethyl methanethiosulfonate indicates that a cysteine at position 334 lies within the outward facing, water-accessible surface of the protein. Login to comment

PMID: 15709975 [PubMed] Riordan JR et al: "Assembly of functional CFTR chloride channels."

No. Sentence Comment

97 This R334C variant exhibited three subconductance states, as does the wild-type, and these states were altered simultaneously and with the same kinetics by a positively charged MTS reagent also making it seem highly unlikely that the segments from more than one CFTR polypeptide come together to form the permeation pathway. Login to comment

PMID: 16191501 [PubMed] Chou LS et al: "A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis transmembrane conductance regulator gene as a model."

No. Sentence Comment

126 For example, in addition to the R334W mutation studied, 6 more mutations have been reported for this amino acid (R334C, E, H, K, L, Q).35 It would be interesting to compare the melting curves of all 7 R334 mutations to see how many can be distinguished from each other. Login to comment

PMID: 16227620 [PubMed] Zhang ZR et al: "State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

2 In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET؉ ), which increases conductance, occurred only during channel closed states. Login to comment
4 R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET؉ . Login to comment
5 Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. Login to comment
6 In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. Login to comment
7 These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. Login to comment
8 The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs. Login to comment
16 In that study, real time modification of single R334C-CFTR channels was observed during patch clamp experiments by the sulfhydryl-modifying agent, MTSETϩ , diffusing to the tip of the electrode; the resulting deposition of positive charge increased the open channel conductance. Login to comment
18 Therefore, we hypothesized that the accessibility or reactivity of the engineered cysteine in R334C-CFTR for modification by MTSETϩ may be favored by the closed state. Login to comment
19 To test this hypothesis, we performed a series of experiments to measure the rate coefficients for modification by MTSETϩ and MTSES- under a variety of conditions that alter the channel open probability (Po) of R334C-CFTR. Login to comment
22 EXPERIMENTAL PROCEDURES Preparation of Oocytes and cRNA-For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers (18). Login to comment
40 All single channel recordings for R334C- and R34C/K1250A-CFTR used asymmetrical [Cl- ] in order to increase the single channel amplitude at VM ϭ -100 mV, where the pipettes were filled with a low [Cl- ]-containing solution (see below). Login to comment
45 R334C-, R334C/ K464A-, and R334C/K1250A-CFTR channels were activated by excision into intracellular solution containing 300 mM NMDG-Cl, 1.1 mM MgCl2, 2 mM Tris-EGTA, 1 mM MgATP, 10 mM TES (pH 7.4), 50 units/ml PKA. Login to comment
46 In experiments designed to increase Po of R334C-CFTR channels, 2.75 mM AMP-PNP was added to intracellular solution containing 1 mM MgATP and 100 units/ml PKA. Login to comment
60 The bin widths for R334C-CFTR recordings in the absence of AMP-PNP were 100 ms, and for R334C-CFTR in the presence of additional AMP-PNP or for a dual mutant R334C/K1250A, bin widths were 500 and 1000 ms, respectively. Login to comment
71 RESULTS R334C Channels Are Modified by MTSETϩ Only in the Closed State-We have shown previously that the MTSETϩ -induced covalent modification of a cysteine engineered at CFTR position 334 (in transmembrane domain 6) increased single channel conductance without altering gating properties (17, 18). Login to comment
75 Despite hours of recording of R334C-CFTR channels, the process of modification was observed to occur only during the closed interval (i.e. sometime between the last opening with lower conductance and the first opening with higher conductance). Login to comment
76 This observation led us to hypothesize that modification of R334C-CFTR might be favored by the closed state. Login to comment
77 To test this hypothesis, we coupled the R334C mutation with a mutation at the Walker lysine of NBD2, K1250A, which prolongs the open burst duration of CFTR channels (21-23). Login to comment
78 Fig. 1B shows a recording of a single R334C/ K1250A-CFTR channel, where the electrode was backfilled with 200 ␮M MTSETϩ . Login to comment
81 Hence, even when Po was increased by the K1250A mutation, modification at R334C did not take place in the open state. Login to comment
82 These observations strongly suggest that modification of R334C-CFTR by MTSETϩ is favored by the closed state. Login to comment
83 Movement in the Outer Vestibule of the CFTR Pore 41998 As shown in Fig. A and reported previously (17), R334C-CFTR channels exhibit stable subconductance behavior, including transitions to s1, s2, and f conductance states. Login to comment
84 In contrast, all of the open bursts of R334C/K1250A-CFTR before modification lacked transitions between conductance states and were "locked" in the s2 state (Fig. 1B). Login to comment
85 Following MTSETϩ -induced modification, R334C/K1250A-CFTR channels opened to, and remained locked in, the s2 state. Login to comment
86 Amplitudes for the s2 state in R334C/K1250A-CFTR were not significantly different from the amplitudes of the s2 state of R334C-CFTR before and after modification (p ϭ 0.85) (17). Login to comment
87 These data suggest that interruption of the ATP-dependent gating cycle leads to stabilization of the pore conformation, resulting in fewer transitions between the three open conductance levels characteristic of R334C-CFTR; this provides further support for the notion that transitions between open conductance levels in CFTR channels are linked to NBD-mediated gating events. Login to comment
88 Macroscopic Kinetics of Modification Were Altered in the Presence of AMP-PNP-If modification of R334C-CFTR is favored by the closed state, we would expect to observe a slowing of the macroscopic time course of modification under conditions that increase Po. Login to comment
89 To test this hypothesis, we studied outside-out macropatches pulled from oocytes expressing R334C-CFTR (17). Login to comment
93 To increase the Po of R334C-CFTR channels, we included a poorly hydrolyzable ATP analogue, AMP-PNP, at 2.75 mM in addition to ATP in the pipette (24, 25). Login to comment
94 Fig. 2B shows that the increase in macroscopic current upon exposure of R334C-CFTR channels to MTSETϩ in the presence of cytosolic ATP ϩ AMP-PNP exhibited a somewhat different time course compared with experiments with ATP alone. Login to comment
97 Hence, modification of R334C-CFTR in the presence of mixtures of ATP and AMP-PNP occurred in two phases. Login to comment
102 To understand better the results from macropatch experiments, we performed detached inside-out single channel recordings in R334C-CFTR, in the presence of ATP ϩ AMP-PNP, using the real time modification approach. Login to comment
108 A, a representative trace for R334C-CFTR in the excised, inside-out patch configuration during real time modification by MTSETϩ backfilled in the pipette. Login to comment
111 Current levels for unmodified R334C-CFTR are indicated by the four dashed lines (in order from top to bottom) c, s1, s2, and f, representing the closed, subconductance 1, subconductance 2, and full conductance states. Login to comment
112 B, representative trace for R334C/K1250A-CFTR under identical conditions. Login to comment
115 Modification of R334C-CFTR is slowed in the presence of AMP-PNP. Login to comment
116 A, a representative recording of macroscopic current of R334C-CFTR. Login to comment
120 B, a representative recording of macroscopic current from R334C-CFTR in the presence of ATP ϩ AMP-PNP. Login to comment
122 Movement in the Outer Vestibule of the CFTR Pore DECEMBER 23, 2005•VOLUME 280•NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 41999 openings and prolonged modified openings induced by AMP-PNP were "locked" in the s state, as was found for R334C/K1250A-CFTR with ATP alone, and the s2 state amplitudes were virtually identical to those for the s2 state of R334C-CFTR in the presence of ATP alone (p Ͼ 0.5). Login to comment
124 We analyzed the mean burst duration of unmodified R334C-CFTR channels either in the presence of ATP alone (Fig. 4A) or in the presence of ATP ϩ AMP-PNP (Fig. 4C). Login to comment
127 In contrast, the histogram for R334C-CFTR channels in the presence of ATP ϩ AMP-PNP was fit best with the sum of two exponential functions having ␶B1 ϭ 0.52 Ϯ 0.16 s (p Ͼ 0.1 compared with ␶B obtained with ATP alone) and ␶B2 ϭ 10.3 Ϯ 1.3 s (Fig. 4D). Login to comment
128 The fractional amplitudes contributing to the fits corresponding to ␶B1 and ␶B2 were 71 and 29%, respectively, which are very similar to the fractional amplitudes for ␶1 and ␶2 obtained from the kinetic analysis of modification of R334C-CFTR macroscopic current by MTSETϩ in the presence of ATP ϩ AMP-PNP (69 Ϯ 4 and 31 Ϯ 4%, respectively). Login to comment
129 These data suggest that the faster phase of macroscopic modification arises from modification of R334C-CFTR channels with burst duration of ϳ0.5 s, and the slower phase of macroscopic modification of R334C-CFTR channels reflects the modification of those channels with burst duration of ϳ10 s. Hence, these resultssuggestthatthemodificationratecoefficientslowsunderconditions thatincreasePo,whichisconsistentwiththenotionthatMTSETϩ -induced modification in R334C-CFTR is favored by the closed state. Login to comment
134 This individual patch contained at least three R334C-CFTR channels, recorded in the presence of ATP plus AMP-PNP. Login to comment
146 We studied outside-out macropatches from oocytes expressing R334C/K1250A- or R334C/K464A-CFTR to determine the effects of these gating domain mutations on the kinetics of modification, using experimental procedures similar to those described above. Login to comment
147 Upon exposure to MTSETϩ , the macroscopic current for R334C/K1250A-CFTR increased rapidly at first, followed by a slower increase in current, reflecting a complicated modification process (Fig. 5A); the kinetics of modification were described best by the sum of two exponential functions. Login to comment
149 In five experiments (TABLE ONE), ␶1 averaged 12.5 Ϯ 0.94 s (fractional amplitude 74.7 Ϯ 1.3%), which was significantly larger than the value of ␶ for R334C-CFTR in the presence of ATP alone and the value of ␶1 for R334C-CFTR in the presence of ATP ϩ AMP-PNP (p Ͻ 0.001). Login to comment
150 For those five experiments, ␶2 averaged 225 Ϯ 29 s (fractional amplitude 25.3 Ϯ 1.3%), which was somewhat larger than the value of ␶2 for R334C-CFTR in the presence of ATP ϩ AMP-PNP (p ϭ 0.049). Login to comment
151 The modification rate coefficients for MTSETϩ in R334C/K1250A-CFTR were 9,840 Ϯ 626 M -1 s-1 and 482 Ϯ 65 M -1 s-1 , respectively. Login to comment
153 In other words, while R334C/K1250A-CFTR channels are closed, they stay closed approximately as long as R334C-CFTR channels do, which provides an opportunity for rapid modification. Login to comment
154 When R334C/K1250A-CFTR channels are open, they typically stay open much longer than R334C-CFTR channels do, which reduces the macroscopic modification rate coefficient. Login to comment
155 These interpretations are supported, at least in part, by the single channel behavior of R334C/K1250A-CFTR. Login to comment
157 The top, middle, and bottom traces are from near the beginning, middle, and end of the experiment, respectively. One can see that R334C/K1250A-CFTR channel openings lack prominent transitions between conductance states, no matter how long the burst duration. Login to comment
159 In a manner similar to the experiments using R334C-CFTR in the presence of ATP ϩ AMP-PNP, the briefer open bursts were always modified earlier than the longer bursts. Login to comment
161 However, we were able to estimate mean burst duration of R334C/K1250A-CFTR channels. Login to comment
163 The fractional amplitudes contributed by ␶B1 and ␶B2 were 77 and 23%, respectively, which are very compatible with the fractional amplitudes for ␶1 (75%) and ␶2 (25%) for the kinetics of macroscopic modification of R334C/K1250A-CFTR. Login to comment
164 This suggests that the faster phase of macroscopic modification of R334C/K1250A-CFTR by MTSETϩ could be attributed to modification of channels with burst duration of ϳ4 s, FIGURE 5. Login to comment
165 MTSET؉ -induced modification of R334C/K1250A-CFTR and R334C/ K464A-CFTR. Login to comment
166 Shown are outside-out macropatches from oocytes expressing either R334C/K1250A-CFTR (A and C) or R334C/K464A-CFTR (B). Login to comment
169 The process of modification of R334C/K1250A-CFTR by MTSETϩ at Vm ϭ ϩ80 mVwasfitbestwiththesumoftwoexponentialfunctions,with␶1 ϭ14.8sand␶2 ϭ158s in this experiment. Login to comment
170 The kinetics of modification of R334C/K1250A-CFTR by MTSETϩ at Vm ϭ -80 mV also were described best by the sum of two exponential functions, having values of ␶1 ϭ 12.9 s and ␶2 ϭ 126 s in this experiment. Login to comment
171 In contrast, the kinetics of modification of R334C/K464A-CFTR by MTSETϩ were fit best with a first-order exponential function, with ␶ ϭ 1.92 s in this experiment. Login to comment
173 Effects of AMP-PNP on mean burst duration of unmodified R334C-CFTR channels. Login to comment
174 A, R334C-CFTR channels recorded with ATP and PKA. Login to comment
177 C, after R334C-CFTR channels were activated by ATP and PKA, 2.75 mM AMP-PNP was added to the solution. Login to comment
186 These results are consistent with our hypothesis that modification of R334C is favored by the closed state. Login to comment
187 We also reasoned that if modification of R334C-CFTR channels is favored by the closed state, the modification rate coefficient should be higher under conditions that reduce Po. Login to comment
188 To test this hypothesis, we first studied outside-out macropatches of R334C-CFTR channels in the presence of 0.2 mM ATP and measured the kinetics of modification. Login to comment
189 Surprisingly, the time constant of modification was identical to that observed for R334C-CFTR in the presence of 1 mM ATP (p ϭ 0.72; TABLE ONE). Login to comment
190 We speculated that 0.2 mM ATP may slightly reduce the Po of R334C-CFTR channels, but not to a degree that could alter significantly the kinetics of modification; indeed, the overall Po of R334C-CFTR channels recorded in the presence of 1 mM ATP is already reduced, compared with WT-CFTR under identical conditions (0.24 Ϯ 0.04 (17) versus 0.34 Ϯ 0.03 (27), respectively). Login to comment
191 Therefore, we recorded from giant outside-out patches pulled from oocytes expressing R334C/ K464A-CFTR, which would reduce Po considerably by prolonging the interburst closed durations (21-23) (Fig. 5B). Login to comment
192 The macroscopic current of R334C/K464A-CFTR was increased rapidly upon application of 10 ␮M MTSETϩ . Login to comment
193 The kinetics of modification were described best by a first-order exponential (TABLE ONE; p ϭ 0.004 compared with ␶ for R334C-CFTR). Login to comment
194 The modification rate coefficient for MTSETϩ in R334C/K464A-CFTR was 41,864 Ϯ 4,229 M -1 s-1 , which is roughly 2-fold higher than that in R334C-CFTR under identical conditions (p ϭ 0.007). Login to comment
196 Kinetics of Modification by MTSES- -Because our previous studies (17, 18) showed that the electrostatic potential in the outer vestibule affects the kinetics of modification at R334C, we asked whether the modification rate coefficient (and its potential state dependence) for a negatively charged thiol-modifying reagent was different from that measured for the positively charged MTSETϩ . Login to comment
197 Fig. 7 shows outside-out macropatch recordings from oocytes expressing either R334C-CFTR or R334C/K1250A-CFTR, with rapid exposure to 50 ␮M MTSES- . Login to comment
198 Macroscopic currents from R334C- and R334C/K1250A-CFTR were decreased upon exposure to MTSES- (due to deposition of negative charge) by 75 Ϯ 6 and 77 Ϯ 5%, respectively. Login to comment
199 The kinetics of modification of R334C-CFTR by MTSES- were fit best with a first-order exponential function (TABLE ONE). Login to comment
200 The macroscopic kinetics of modification of R334C/K1250A-CFTR were fit best with the sum of two exponential functions (TABLE ONE; the fractional amplitudes were 84 Ϯ 2.7% for ␶1 and 16 Ϯ 2.7% for ␶2), as was found for MTSETϩ . Login to comment
201 The modification rate coefficients for MTSES- in both R334C-CFTR and R334C/K1250A-CFTR were Ͼ3-fold lower than those measured for MTSETϩ in the same mutants (p Ͻ 0.001). Login to comment
203 More importantly, the kinetics of modification of the engineered cysteine at R334C by MTSES- were state-dependent, as described above for modification by MTSETϩ . Login to comment
206 Upon rapid exposure to MTSETϩ , macroscopic inward current was increased, reflecting modification of R334C/ K1250A-CFTR channels. Login to comment
208 Kinetics of modification of single R334C/K1250A-CFTR channels. Login to comment
217 All openings of R334C/K1250A-CFTR channels exhibited conductance equivalent to the s2 conductance state of R334C-CFTR. Login to comment
220 MTSES- -induced modification of R334C-CFTR and R334C/K1250A-CFTR. Login to comment
221 Outside-out macropatches were pulled from oocytes expressing either R334C-CFTR or R334C/K1250A-CFTR. Login to comment
223 A, R334C-CFTR channels were activated by ATP plus PKA. Login to comment
225 The dashed line indicates a fit of the data to a first-order exponentialfunction,having␶ϭ4.5sinthisexperiment.B,arepresentativerecordingof macroscopic current of R334C/K1250A-CFTR under identical conditions. Login to comment
229 These results indicate that the direction of anion movement does not affect the rate of modification by MTS reagent at R334C. Login to comment
231 Single R334C-CFTR channels studied using real time modification only showed a reaction to MTSETϩ during a closed state, even when channel Po was increased dramatically by exposure to mixtures of ATP and AMP-PNP or by the addition of the Walker A mutation K1250A. Login to comment
232 Macropatch currents recorded from oocytes expressing R334C-CFTR increased rapidly upon abrupt exposure to MTSETϩ (or decreased rapidly upon abrupt exposure to MTSES- ). Login to comment
233 Under conditions that increase channel activity (i.e. R334C-CFTR with ATP ϩ AMP-PNP, or R334C/K1250A-CFTR with ATP alone), the kinetics of modification were slowed. Login to comment
234 Under conditions that decrease channel activity (R334C/K464A-CFTR), the rate of modification was increased dramatically. Login to comment
237 Our data indicate that the rate of covalent modification at R334C differs dramatically between closed and open channels. Login to comment
239 However, we previously found that the macroscopic conductance of whole oocytes expressing R334C-CFTR channels was sensitive to bath pH, due to titration of the partial negative charge on the unmodified cysteine (17, 18). Login to comment
240 This observation suggests that R334C indeed faces the water-soluble pore while the channels are open, because protons can access this residue. Login to comment
241 Hence, the state-dependent ability of MTS reagents to interact with the engineered cysteine of R334C-CFTR most likely reflects a difference in the reactivity of that cysteine during channel closure rather than physical obstruction that reduces accessibility. Login to comment
243 Hence, we cannot say that R334C changes its position between open and closed states but rather must limit ourselves to saying that the orientation of R334C relative to the other residue or the distance between them changes as a function of channel gating. Login to comment
244 Nonetheless, these data suggest that changes in the rate coefficients for thiol-modifying reagents at R334C under different experimental conditions reflect conformational changes in the outer vestibule of the CFTR pore, which are associated with ATP-dependent gating. Login to comment
246 As described previously (17), channels formed by WT-CFTR and many pore domain mutants, including R334C-CFTR, exhibit two subconductance states (s1 and s2) as well as the full conductance state (f). Login to comment
248 In R334C-CFTR channels, the most stable conducting state is the s2 state, whereas in WT-CFTR channels, the most stable conducting state is the f state (17). Login to comment
249 Results from the present study show that when R334C-CFTR channels are locked open by either AMP-PNP or the addition of the K1250A mutation, they are locked into the s2 state. Login to comment
253 R334C-CFTR channels almost always transition briefly to the f state before closure (see the arrowheads in Fig. 3) (17); the f state may represent an unstable conformation that serves as a transition intermediate between the stable s2 state and the stable c state. Login to comment

PMID: 17673962 [PubMed] Zhou JJ et al: "Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

73 This is consistent with previously reported weakening of intracellular Pt(NO2)4 2À block in R334C (Gong & Linsdell, 2003b). Login to comment
85 Figure 3 shows the blocking effects of internally applied Pt(NO2)4 2À in six different channel mutants (R334C, R334E, R334H, R334K, R334L, R334Q) under conditions of both low (Fig. 3a) and high (Fig. 3b) extracellular ClÀ concentration. Login to comment
90 With low extracellular ClÀ concentrations, the Kd for Pt(NO2)4 2À block (at 0 mV) was significantly increased in all six R334 mutants studied (Fig. 5a), although it is clear that R334C and R334E had far greater effects on Kd compared to other amino acid substitutions. Login to comment
91 With elevated extracellular ClÀ , the Kd(0) was significantly increased only in R334C and R334E; not significantly altered in R334K, R334L and R334Q; and significantly decreased in R334H (Fig. 5b). Login to comment
93 These R334 mutations also exhibited a weakened sensitivity of blocker voltage dependence (quantified as -zd, Fig. 5) to external ClÀ concentration (Fig. 5c), although because of the small magnitude of this effect only R334C, R334H and R334Q reached a level of statistical significance (Fig. 5c). Login to comment
106 Comparison of the mean Kd estimated for suramin (at 0 mV) shows that R334C, R334E, R334K, R334L and R334Q were all associated with weakened suramin block, with only R334H failing to significantly affect suramin block (Fig. 7). Login to comment
107 Suramin block was particularly weakened in R334C and R334E (Figs. 6, 7) such that the profiles of mutation effects on block by internal Pt(NO2)4 2À and suramin are very similar. Login to comment
129 Mean of data from three to eight patches. Fitted lines are to equation 1 as described in Figure 1 for wild type and R334Q and with the following parameters for other channel variants: R334C 4 mM external ClÀ , Kd(0) = 1362 lM, zd = À0.295; R334C 154 mM external ClÀ , Kd(0) = 836 lM, zd = À0.219; R334E 4 mM external ClÀ , Kd(0) = 759 lM, zd = À0.376; R334E 154 mM external ClÀ , Kd(0) = 564 lM, zd = À0.173; R334H 4 mM external ClÀ , Kd(0) = 140 lM, zd = À0.166; R334H 154 mM external ClÀ , Kd(0) = 119 lM, zd = À0.149; R334K 4 mM external ClÀ , Kd(0) = 143 lM, zd = À0.314; R334K 154 mM external ClÀ , Kd(0) = 317 lM, zd = À0.374; R334L 4 mM external ClÀ , Kd(0) = 176 lM, zd = À0.258; R334L 154 mM external ClÀ , Kd(0) = 284 lM, zd = À0.366 extracellular Pt(NO2)4 2À by normalizing current amplitude at the hyperpolarized extreme of the voltage range studied, -80 mV (Fig. 10b). Login to comment
159 These plots represent mean data from four to seven patches. Fitted lines are to equation 1 with the following parameters: wild type, Kd(0) = 2.51 lM, zd = À0.042; R334C, Kd(0) = 18.5 lM, zd = À0.056; R334E, Kd(0) = 25.0 lM, zd = À0.107; R334H, Kd(0) = 3.10 lM, zd = À0.085; R334K, Kd(0) = 6.31 lM, zd = À0.232; R334L, Kd(0) = 4.08 lM, zd = À0.061; R334Q, Kd(0) = 6.64 lM, zd = À0.239 with our previous suggestion that intracellular Au(CN)2 À blocks the channel by interacting directly with R334, several reasons prompt us to suggest that Pt(NO2)4 2À does not interact directly with the arginine side chain at this position. Login to comment
162 Thus, while Pt(NO2)4 2À block is particularly weak in R334C and R334E (Figs. 3-5), there is no strong correlation between the apparent affinity of Pt(NO2)4 2À block and the nature of the side chain present at position 334. Login to comment
166 The similar effects of mutations on block by intracellular suramin and intracellular Pt(NO2)4 2À - in particular, the strong effects of R334C and R334E on the inhibitory effects of both blockers - suggest that these mutations affect suramin block and Pt(NO2)4 2À block by a common mechanism. Login to comment
221 However, disruption of the effects of intracellular blockers is particularly pronounced in R334C and R334E (Figs. 5, 7). Login to comment
222 Currently, we have no explanation as to why R334C and R334E have so much more dramatic effects on block by intracellular Pt(NO2)4 2À and suramin than other R334 mutations. Login to comment
227 b,c Mean fraction of control normalized current remaining in the presence of 10 mM extracellular Pt(NO2)4 2À at different membrane potentials for different channel variants: b wild type (d), R334C (. Login to comment

PMID: 17849169 [PubMed] Liu X et al: "A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator."

No. Sentence Comment

119 Similar behavior was observed in R334C CFTR (Fig. 6B). Login to comment
152 (B) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), an oocyte expressing R334C CFTR was exposed to: 1 mM MTSET+ (black circles), 100 lM GSH and 10 mM GSH (open triagles) could, in principle, remove bound copper from the adventitious site. Login to comment

PMID: 18056267 [PubMed] Beck EJ et al: "Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating."

No. Sentence Comment

85 Application of Cd2ϩ to activated R334C CFTR reduced whole cell Cl-conductance by Ͼ80%, with the inhibition completely reversible upon Cd2ϩ removal (Fig. 1C). Login to comment
93 Both Cd2ϩ and MTSEA had significant effects on the conductances of only five (I331C, L333C, R334C, K335C, and T338C) of the 26 Cys-substituted channels examined. Login to comment
100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
104 The region highlighted in red indicates the putative location of theresiduesanalyzed.B,andC,typicalrecordingofwholecellconductanceof oocytes expressing WT (B) or R334C-CFTR (C). Login to comment
115 An example of this protocol applied to R334C-CFTR expressing oocytes is shown in Fig. 2A. Login to comment
117 The magnitude of the loss of Cd2ϩ sensitivity was similar to that observed for activated R334C-CFTR that had been modified by MTSEA (e.g. Fig. 1C). Login to comment
127 For example, whereas L333C in the Glu1371 (WT) channel was inhibited by either Cd2ϩ or MTSEA, neither reagent was particularly effective when this mutation was present in the Gln1371 background 200 150 100 50 0 µS 15001000500 s IBMX Cd 2+ MTSEA DTT -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C Cd 2+ aM Cd 2+ bM Cd 2+ uM A B FIGURE 2. Login to comment
129 A, representative experiment in which MTSEA (1 mM) was applied for 3 min to R334C-CFTR channels that are closed. Login to comment
131 B, summary of effects of Cd2ϩ on MTSEA-modified I331C, L333C, R334C, K335C, and T338C channels. Login to comment
135 MTSEA 1371Q 600 400 200 µS 200150100500 s Cd 2+ 1371E -40 0 40 % Change in conductance I331C L333C R334C K335C T338C 1371E 1371Q * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * 1371Q 800 600 400 µS 2001000 s MTSEA 1371E B D E 1 2 30 s1 pAWT; Po=0.18 A 3 1 2 100 s1 pAE1371Q; Po=0.94 C FIGURE 3. Login to comment
152 In contrast, the pore-lining residues R334C and T338C exhibited very small or no differences in their functional effects in the Glu1371 and Gln1371 channels, respectively. Login to comment
153 The differences between Glu1371 and Gln1371 backgrounds in the effects of Cd2ϩ and MTSEA on I331C, L333C, R334C, K335C, and T338C channels are summarized in Fig. 3 (C and E), respectively. Login to comment
158 The cysteine residues R334C and T338C, postulated to be pore-lining residues, showed no changes in their rates of modification by either MTSEA or MTSES. Login to comment
179 For example, under minimally active conditions, the stimulatory effect of MTSEA on R334C and K335C conductance was greater than under maximally active conditions. Login to comment
180 MTSES, however, had a smaller inhibitory effect on R334C and K335C when minimally activated. Login to comment
184 Under minimal activation conditions (0.02 mM IBMX), the cysteine residues R334C, K335C, and T338C showed no significant differences in their modification rates by either MTSEA or MTSES (Fig. 6). Login to comment
197 Kinetic analyses of channel gating revealed that the decrease in open probability of MTSET-modified I331C and L333C channels was primarily because of an increase in the mean interburst duration of the A B 1.00.50.0 G0.02/ G1 I331C L333C R334C K335C T338C 200 100 0 µS 8006004002000 s 0.02 1 IBMX (mM) C -100 100 % Change in conductance I331C L333C R334C K335C T338C 0.02 mM IBMX 1 mM IBMX * * * * -80 -60 -40 -20 0 % Change in conductance I331C L333C R334C K335C T338C * * * MTSEA MTSES FIGURE5.EffectsofMTSEA,andMTSESdependonCFTRactivationlevels. Login to comment
216 Although both studies identified I331C, L333C, R334C, and K335C as accessible to MTS reagents, we find that MTSEA increased the conductance of R334C- and K335C-expressing oocytes, whereas it was reported in the previous study to decrease channel currents. Login to comment
220 However, our observations on the accessibility of R334C, K335C, and T338C and the inaccessibility of R347C are consistent with other studies (10, 11). Login to comment
223 It is possible that this mutation rather than the open 150 125 100 %G/Gi 600 s K335C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C 100 50 %G/Gi 3002001000 s I-1.0; 100 µM I-0.02;10 µM MTSEA I331CL333CR334CK335CT338C 100 75 50 25 0 %G/Gi 180120600 s I-0.02; 10 µM I-1.0; 10 µM 200 150 100 %G/Gi 120600 s I-0.02; 10 µM I-1.0; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 80 60 %G/Gi 9060300 s K335C I-1.0; 10 µM I-0.02; 10 µM 100 50 %G/Gi 180120600 s T338C I-1.0; 10 µM I-0.02; 10 µM 10 1 10 2 10 3 10 4 Modification rate (M -1 s -1 ) I331C L333C R334C K335C T338C MTSES 100 75 50 25 %G/Gi 120600 s I-1.0; 10 µM I-0.02; 10 µM 100 75 50 %G/Gi 3602401200 s I-1.0; 100 µM I-0.02; 10 µM 100 75 %G/Gi 180120600 s I-0.02; 100 µM I-1.0; 1 mM A B FIGURE 6. Login to comment
239 Furthermore, the pore-lining residues R334C and T338C showed no state-dependent changes in reactivity, which also suggests that there are no significant changes in the local electrostatic potential during channel gating. Login to comment
242 Hence, a small fraction of the increased reactivity of I331C, and L333C at low IBMX concentrations could be due to a relief from this block, although such an increase in reactivity is not observed for R334C and T338C. Login to comment
245 Zhang et al. (26) reported that the rate of MTSET modification of R334C-CFTR expressed in oocytes was monotonic in the WT background but followed a bi-exponential decay because of an additional slower (nearly 20 times slower) component in the K1250A background. Login to comment
247 In contrast, our results show that the reactivity of R334C does not exhibit state dependence either under varying activation levels or in the E1371Q channel. Login to comment

PMID: 18167343 [PubMed] Fatehi M et al: "State-dependent access of anions to the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

59 As shown previously (9, 15), the R334C mutation itself induced inward rectification because of removal of the native positive charge and reduced electrostatic attraction of extracellular Cl- ions. Login to comment
64 In contrast to these results obtained with inclusion of MTS reagents in the pipette solution, we found that MTSES was apparently unable to modify R334C using a pretreatment protocol. Login to comment
66 This lack of effect could reflect that MTSES does not covalently label R334C and so must be present during the experiment to exert its effect, that covalent modification does occur but is relatively transient, or that covalent modification takes place during pipette application but not during pretreatment. Login to comment
69 Following cAMP stimulation, MTSES was able to modify R334C channels and induce increased inward rectification of the I-V relationship (Fig. 1, G and H), indicating that stable covalent modification of this introduced cysteine could take place but was somehow dependent on the activation state of the channels. Login to comment
71 Conformational Change in the Pore on Activation of CFTR MARCH 7, 2008•VOLUME 283•NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6103 Next we asked if this apparent state-dependent access of MTSES was limited to R334C or if a similar effect could be seen with cysteines introduced at other sites in the pore. Login to comment
74 In fact, similar charge-dependent effects were observed in R334C, K335C, T338C, and S341C (Fig. 3). Login to comment
77 The state dependence of modification of cysteines introduced at these sites was studied using the MTS pretreatment protocol described above for R334C. Login to comment
79 As with R334C (Fig. 1), MTSES induced inward rectification of the I-V relationship (indicating covalent modification of the substituted cysteine) when included in the pipette solution but not when preincubated with the cells. Login to comment
82 Modification of R334C-CFTR by external MTS reagents. Login to comment
86 C, the same experimental protocol in R334C-CFTR illustrates modification by MTSET and MTSES, seen clearly in the IREL-V relationships as changes in current rectification. Login to comment
95 Two positively charged maleimide derivatives, MBTA and TMAEM, had similar effects on R334C as those observed with MTSET (Fig. 5), leading to a more linear I-V relationship. Login to comment
100 The effects of MTSES on R334C were mimicked by another negatively charged reagent, pCMBS (Fig. 6). Login to comment
114 F, wild type (both panels); E, R334C (left); Ⅺ, K335C (left); ‚, F337C (right); ƒ, T338C (right); छ, S341C (right) (mean of data from 3-9 patches). Login to comment
136 Positively charged maleimide derivatives modify R334C-CFTR prior to channel activation. Login to comment
140 Conformational Change in the Pore on Activation of CFTR 6106 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 10•MARCH 7, each of R334C, K335C, and S341C, like T338C, the apparent degree of Au(CN)2 - modification as determined by the KCN- sensitive component of the current was significantly enhanced by cAMP stimulation (Fig. 7E). Login to comment
155 In contrast, MTSES cannot even modify R334C in the outer mouth of the pore under these conditions. Login to comment
157 Reactivity of external MTSET and MTSES with R334C-CFTR has been studied previously, and it was shown that following channel activation both of these reagents modified this introduced cysteine only when the channel was closed (18). Login to comment
162 State-dependent modification of R334C-CFTR by external pCMBS. Login to comment
171 This gating cycle also appears to involve changes in accessibility to both MTSET and MTSES, at least as reported by a cysteine substituted for Arg-334 (18), although in this case there is no apparent charge dependence of accessibility. Login to comment
188 D, an example I-V relationship for R334C pretreated with 10 ␮M KAu(CN)2 plus forskolin (10 ␮M) and IBMX (100 ␮M) for 1 min recorded before (control) and after treatment with 50 ␮M KCN is shown. Login to comment
189 E, the mean change in CFTR macroscopic conductance for R334C, K335C, F337C, and S341C following addition of KCN without (white bars) or with (black bars) cAMP pretreatment is shown (mean of data from 4-5 patches). Login to comment
192 Because modification of R334C, which is thought to be located at the outer mouth of the pore, is state-dependent, we suggest that the conformational change associated with channel activation must affect the very outermost part of the pore to influence access of external ions to Arg-334. Login to comment

PMID: 18421494 [PubMed] Cui G et al: "Mutations at arginine 352 alter the pore architecture of CFTR."

No. Sentence Comment

105 Transitions to these subconductance levels occur rarely in WT-CFTR but more frequently in some pore-domain mutants, such as R334C and T338A, although the relative conductances between levels s1, s2, and f are maintained (Zhang et al. 2005a). Login to comment
114 In contrast, our previous experiments (Zhang et al. 2005a) with R334C-CFTR showed a more regular transition pattern: Within nearly every burst there were transitions to all three conductance states, with most bursts ending in the f state. Login to comment

PMID: 18449561 [PubMed] Zhou JJ et al: "Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore."

No. Sentence Comment

101 This suggests that both positively and negatively charged MTS reagents can modify both R104C and R117C independently of the state of channel activation, a situation that contrasts with R334C, K335C, and other TM6 mutants. Login to comment

PMID: 19754156 [PubMed] Alexander C et al: "Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore."

No. Sentence Comment

52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment

PMID: 20805575 [PubMed] Bai Y et al: "Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation."

No. Sentence Comment

82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
83 The single-channel amplitude is unsolv- able in the cases of R334C, S341C, R347C, and R352C due to a limited bandwidth, whereas it is 0.2-0.3 pA for Data analysis Current traces containing fewer than three channel opening levels and lasting for >1 min were selected for single-channel kinetic analysis using a program developed by L. Csanády (2000). Login to comment

PMID: 9922376 [PubMed] Dawson DC et al: "CFTR: mechanism of anion conduction."

No. Sentence Comment

480 Three residues, R334C, R347C, neered cysteines can react with the charged MTS reagents in the pore interior. The potential flaw in this assumptionand R352C, were also inhibited by the larger cation MTSET0 . Login to comment

PMID: 22923500 [PubMed] Norimatsu Y et al: "Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator."

No. Sentence Comment

125 Figure 4 shows the time courses for the reactions of [Au(CN)2]- and MTSET+ with R334C CFTR channels. Login to comment
130 Beck et al., (2008) also studied R334C CFTR channels expressed in Xenopus oocytes using a protocol similar to that employed here, but failed to detect increased reactivity toward externally-applied MTSEA+ in the activated state. Login to comment
183 The substantially higher apparent affinity of F342A CFTR for GlyH-101 implies a dramatic slowing of the off-rate for the bound blocker. Login to comment
225 The MsbA-based model of Mornon et al., (2009) also predicts that the side chain of R334 protrudes into the external aqueous environment, and when R334 is mutated to a cysteine in the MsbA-based model of Mornon et al., (2009) using Maestro (version 9.1, Schrödinger LLC), the reactive thiolate is clearly accessible from the extracellular solution (Figure 9C), consistent with the closed state reactivity of R334C observed in the current study and by Zhang et al., (2005). Login to comment
226 On the other hand, the mechanism that renders R334C CFTR unreactive in the conducting state of CFTR is not clear. Login to comment
228 The R334C mutation may cause a significant conformational change to the CFTR protein (not captured in our MD simulation), as suggested by Zhou et al., (2007), that renders the engineered cysteine inaccessible in the conducting state of CFTR. Login to comment
188 Beck et al. (2008) studied R334C CFTR channels expressed in X. laevis oocytes by using a protocol similar to that used here, but they failed to detect increased reactivity toward externally applied 2-aminoethylmethanethiosulfonate in the activated state. Login to comment
196 Time courses of reactions of R334C (an engineered cysteine at position 334) with 10 òe;M [Au(CN)2]afa; (A) or 250 nM MTSETaf9; (B). Login to comment
201 For both [Au(CN)2]afa; and MTSETaf9; , the reaction rate for the R334C CFTR before activation of the channels was faster than the rate after activation. Login to comment
356 Cells expressing the R334C CFTR were preincubated with MTSESafa; with and without an activating cocktail of forskolin and IBMX, and the reaction, which was inferred on the basis of changes in the rectification ratio of the CFTR I-V curve, was observed only with cells exposed to the activating cocktail. Login to comment
368 with Maestro 9.1 (Schro &#a8;dinger) in the MsbA-based model, the reactive thiolate is clearly accessible from the extracellular solution (Fig. 9C), which is consistent with the closed-state reactivity of R334C observed in the current study and in the study by Zhang et al. (2005). Login to comment
369 The mechanism that renders the R334C CFTR unreactive in the conducting state is not clear. Login to comment
371 The R334C mutation may cause significant conformational changes in the CFTR protein (that are not captured in our MD simulation), as suggested by Zhou et al. (2007), which render the engineered cysteine inaccessible in the conducting state of the CFTR. Login to comment

PMID: 22680785 [PubMed] Liu X et al: "Thermal instability of DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) channel function: protection by single suppressor mutations and inhibiting channel activity."

No. Sentence Comment

74 Additional covalent labeling experiments conducted with R334C/ΔF508 CFTR channels indicated that potentiation of the thermally inactivated conductance at 22 °C by P2 was not attributable to the addition of new channels to the membrane and must therefore reflect a P2-induced increase in channel open probability consistent with that reported by Pedemonte et al.36 (see Supporting text and Figure S2). Login to comment
285 ■ ASSOCIATED CONTENT *S Supporting Information Supporting text and Figures S1 and S2 document the lack of major insertion of new, unlabeled R334C/ΔF508 CFTR channels to the membrane during cAMP stimulation, recovery following warming (Figure S1) and potentiation by PG-01 (P2) following warming (Figure S2). Login to comment

PMID: 22303012 [PubMed] Wang W et al: "Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)."

No. Sentence Comment

145 Zhang et al. (25) showed that R334C, located slightly closer to the outside of TM6 than Thr-338, was modified by extracellular MTS reagents only in the closed state. Login to comment
147 However, these cysteines in the outer pore region (L333C, R334C, and K335C) are not modified by intracellular MTS reagents under any conditions (17, 27), suggesting that unlike T338C they cannot move to a position that is accessible to large cytoplasmic substances. Login to comment
151 For example, L102C in TM1 is modified by internal, but not external MTS reagents (18), a result confirmed by the present results (Figs. 1 and 3), whereas R104C, only 2 residues closer to the external end of TM1, is modified by external, but not internal MTS reagents (28). Login to comment
153 Rate of modification of T338C by external MTSES. Login to comment

PMID: 22160394 [PubMed] Cui G et al: "Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers."

No. Sentence Comment

135 Also, we reported that R334A and R334C exhibited multiple single-channel conductance levels, including subconductance 1 (s1), subconductance 2 (s2), and full conductance states (f). Login to comment
136 R334C can be modified by methanethiosulfonate reagents in a state-dependent manner suggesting that its position moves during channel gating [46]. Login to comment

PMID: 22014307 [PubMed] Liu X et al: "Cystic fibrosis transmembrane conductance regulator: temperature-dependent cysteine reactivity suggests different stable conformers of the conduction pathway."

No. Sentence Comment

81 Increased temperature altered the rate of modification of R334C CFTR by MTSES- . Login to comment
82 An oocyte expressing R334C CFTR was activated using a stimulatory cocktail and then (A) exposed to 1 mM 2-ME (white bar and circles) and 3 μM MTSES- (dark gray bar and circles) at room temperature. Login to comment
85 Figures 2-4 contain the results of similar experiments conducted with oocytes expressing R334C, I336C, and T338C CFTR channels. Login to comment
97 Temperature Dependence of MTSES- Modification kMTSES (M-1 s-1 ) mutant 22 °C 30 °C 32 °C 37 °C Ea (kJ/mol) R334C 2648 ± 259 (n = 3) 9411 ± 1210 (n = 5) 18407 ± 3240 (n = 3) 98 I336C 1.2a 2.3 ± 0.1 (n = 3) 6.9 ± 0.4 (n = 4) 88 F337C 2.6 ± 0.7 (27 °C)b (n = 3) 5.1 ± 1.2 (n = 3) 19.4 ± 4.4 (n = 4) 157 T338C 4067 ± 573 (n = 5) 7192 ± 370 (n = 4) 7972 ± 1019 (n = 6) 35 a Value from ref 10. b The reaction rate was undetectable at 22 °C, so the value determined at 27 °C was used. Login to comment
106 Arrhenius plots for (A) R334C, (B) I336C, (C) F337C, and (D) T338C CFTR. Login to comment

PMID: 21461971 [PubMed] Krasilnikov OV et al: "ATP hydrolysis-dependent asymmetry of the conformation of CFTR channel pore."

No. Sentence Comment

201 In contrast, real time measurements of covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate indicated that a single CFTR polypeptide forms a CFTR channel with a single pore [25]. Login to comment

PMID: 15361410 [PubMed] Liu X et al: "CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore."

No. Sentence Comment

72 Finally, preliminary experiments indicated that, in contrast to results obtained previously with R334C CFTR, reaction of T338C channels with MTSET1 actually reduced single-channel conductance, rendering the resulting single-channel currents more difficult to discriminate from the small (0.1-0.3 pA) background Clÿ channels sometimes seen in patches from oocytes. Login to comment
94 For ease of comparison, in T338C/R334X (X ¼ A or E) CFTRs and T338H/R334C CFTRs in which the cysteine was always blocked by reaction with MTS reagents or NEM, the titration curves were expressed in a normalized form. Login to comment
104 In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET1 or MTSESÿ , or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Login to comment
177 This procedure attributed the effects of MTSET1 and MTSESÿ on the conductance of R334C CFTR to a change in Co of 45 mV and ÿ30 mV, respectively, a prediction somewhat different from that reported by Smith et al., 2001, in which the effects of MTSET1 and MTSESÿ were attributed to a change in Co of 50 mV and ÿ10 mV, respectively.) Login to comment
190 To test the generality of titration results obtained with T338C CFTR, we compared the titration behavior of T338H CFTR with that of a double mutant, T338H/R334C, in which it was possible to change the charge at position 334 by means of chemical modification. Login to comment
191 Summarized in Fig. 8 B are the results obtained from oocytes expressing either T338H or chemically modified T338H/R334C CFTR (n ¼ 3-4 for each mutant). Login to comment
192 Shown are sample titration curves for T338H and T338H/R334C CFTRs in which the cysteine was modified by MTSET1 , MTSESÿ , and NEM (neutral). Login to comment
195 Similarly, the conductance due to MTSET1 -modified T338H/R334C CFTR was not very sensitive to pH titration. Login to comment
208 NEM- or MTSESÿ -modified T338H/R334C CFTR were more titratable and the apparent pKa values were shifted toward more basic values. Login to comment
209 Because the range of pH values tolerated by oocytes was limited, the apparent pKa values could not be estimated accurately for T338H and T338H/ R334C variants. Login to comment
221 FIGURE 8 The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. Login to comment
222 (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment
225 (B) Sample titration curves for T338H (open triangle) and T338H/R334C CFTR modified by MTSET1 (solid circles), MTSESÿ (open circles), and NEM (shaded triangles). Login to comment
290 In previous experiments using R334C CFTR (Smith et al., 2001) we showed that, in general, the total charge change due to covalent modification was the sum of the charge added by the deposited group and the pH-dependent charge on the thiolate that is neutralized in the formation of a mixed disulfide bond. Login to comment
291 The results shown in Fig. 12 compare the effectsof covalent modificationof T338C CFTR at pH 6 and pH 9, values chosen such that the partial charge on the cysteine thiolate would vary from near zero (pH 6) to near ÿ1 (pH 9). Login to comment
302 The blocking effect of MTSET1 at position 338 contrasts to the effect of the same reagent at position 334, where MTSET1 increased the conductance of R334C CFTR by ;45% (616) at pH 6 and by ;92% (612) at pH 9 (Smith et al., 2001). Login to comment
313 The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment
105 In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET1 or MTSES , or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Login to comment
178 This procedure attributed the effects of MTSET1 and MTSES on the conductance of R334C CFTR to a change in Co of 45 mV and 30 mV, respectively, a prediction somewhat different from that reported by Smith et al., 2001, in which the effects of MTSET1 and MTSES were attributed to a change in Co of 50 mV and 10 mV, respectively.) Login to comment
193 Shown are sample titration curves for T338H and T338H/R334C CFTRs in which the cysteine was modified by MTSET1 , MTSES , and NEM (neutral). Login to comment
196 Similarly, the conductance due to MTSET1 -modified T338H/R334C CFTR was not very sensitive to pH titration. Login to comment
210 Because the range of pH values tolerated by oocytes was limited, the apparent pKa values could not be estimated accurately for T338H and T338H/ R334C variants. Login to comment
226 (B) Sample titration curves for T338H (open triangle) and T338H/R334C CFTR modified by MTSET1 (solid circles), MTSES (open circles), and NEM (shaded triangles). Login to comment
304 The blocking effect of MTSET1 at position 338 contrasts to the effect of the same reagent at position 334, where MTSET1 increased the conductance of R334C CFTR by ;45% (616) at pH 6 and by ;92% (612) at pH 9 (Smith et al., 2001). Login to comment
315 The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment

PMID: 9089437 [PubMed] Cheung M et al: "Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel."

No. Sentence Comment

107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment

PMID: 8744306 [PubMed] Cheung M et al: "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment."

No. Sentence Comment

91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
109 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
116 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment
117 One-minute and 8-min applications of 1 mM MTSET+ inhibited the CFTIR-mediated current of the mutants R334C by 53 ± 6% and 52 ± 7% (n = 3); R347C by 44 ± 2% and 36 + 3% (n = 3) (Fig. 3 D); and R352C by 46 ± 11% and 54.6 ± 10.5% (n = 3). Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
108 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
115 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment

PMID: 15130785 [PubMed] Gong X et al: "Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions."

No. Sentence Comment

35 Results and discussion Previously we characterized the properties of six different R334 mutants (R334C, R334E, R334H, R334K, R334L, and R334Q) [19]. Login to comment
51 Others have previously shown that both R334C [20] and R334W [24] are associated with reduced unitary conductance. Login to comment
65 (A) Unitary current-voltage relationships for each of the channel variants shown in Fig. 1: (d) wild type, (r) R334C, (j) R334E, (}) R334H, (s) R334K, () R334L, (.) Login to comment

PMID: 23709221 [PubMed] Cui G et al: "Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function."

No. Sentence Comment

25 For example,R334C-CFTRchannelsroutinelyopentothes1andthen s2statesandtransitiontothefstatejustbeforeclosing(15).Hence, it is reasonable to believe that CFTR channel pore opening might involve a complicated sequence of multiple steps leading to the occupancy of a stable, full open state. Login to comment

PMID: 23784545 [PubMed] Cai Z et al: "Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms."

No. Sentence Comment

251 The occurrence of subconductance states is more frequent in some site-directed mutations in the MSDs [e.g., R334C-CFTR (70), R347E-CFTR (11), and R352E-CFTR (13)], while wild-type murine CFTR resides for prolonged periods in a minuscule subconductance state and only transitions infrequently to the full open-state (37). Login to comment

PMID: 23955087 [PubMed] Wang W et al: "Relative contribution of different transmembrane segments to the CFTR chloride channel pore."

No. Sentence Comment

187 The proposed relative alignment of TMs 6 and 11 presented in Fig. 9a is consistent not only with the pattern of MTSES accessibility from different sides of the membrane, but also with previous data showing that disulfide bonds can form between T338C and S1118C, and between R334C and T1122C, in open channels [43]. Login to comment

PMID: 24086355 [PubMed] Rahman KS et al: "Modeling the conformational changes underlying channel opening in CFTR."

No. Sentence Comment

65 State-Dependent Accessibility of R334C The arginine at position 334 on TM6 lies at the outer mouth of the pore, and has been suggested to play a role in attracting anions [33,34]. Login to comment
66 It has been observed, however, that modification of cysteine mutants at this site (R334C) occurs only in the closed state, with the site apparently being inaccessible when the channel is open [51-53]. Login to comment
68 In an open state O-CFTR model in which the arginine at position 334 was mutated to a cysteine in silico, R334C is buried by many of its neighboring residues in ECL3 and is found to have a fractional solvent-accessible surface area (SASA) of only 3.8%. Login to comment
69 By comparison, the C0-CFTR model presents R334C in a relatively exposed conformation, with 32.9% of its surface area accessible to solvent. Login to comment
70 This increased accessibility may translate to the higher observed rates of reaction of R334C with extracellular sulfhydryl-modifying reagents in the closed state. Login to comment
97 State dependent-accessibility of R334C. Login to comment
98 These images show CFTR from the extracellular side, with the mutant residue R334C represented in CPK colors, and all residues with atoms within 5 A da; of R334C shown as pink spheres. Login to comment
99 R334C is largely buried by its neighboring residues in the open channel state, exposing only 3.8% of its surface area to solvent. On the other hand, in the closed channel, R334C is more exposed (32.9% accessible). Login to comment
100 This is in accordance with experimental results demonstrating the preferential closed-state accessibility of R334C [51,52]. Login to comment
144 To provide experimental confirmation of the closed and open structures, and the transitions between the two, we asked whether cysteines engineered at these two positions in the double-mutant, R334C/E217C-CFTR, could be functionally crosslinked. Login to comment
146 The traces in Figure 7 show that R334C/E217C-CFTR can repeatedly be activated by stimulation of the co-expressed beta2-adrenergic receptor using isoproterenol, without substantial decrement in peak current prior to exposure to the crosslinker MTS-2-MTS. Login to comment
147 When the same cell was exposed to MTS-2-MTS in the absence of isoproterenol (Figure 7B), when most of the channels should be closed, subsequent exposure to isoproterenol failed to activate CFTR channels to the same degree as prior to MTS-2-MTS; these results are consistent with the notion that R334C and E217C are positioned very near each other in the channel closed state. Login to comment
152 In control experiments, exposure to MTS-2-MTS did not have similar effects on the single mutants R334C-CFTR and E217C-CFTR, with respect to the ability to re-open channels after MTS-2-MTS exposure (Figure S6). Login to comment
154 Inspection of the C0- and O-CFTR models indicates that the side chains of R334C and E217C are, indeed, close enough to be crosslinked by MTS-2-MTS only in the closed state (Figure S8). Login to comment
173 Crosslinking R334C to E217C locks CFTR channels into the closed state. Login to comment
176 B: Representative trace (left) and summary data (right) for macroscopic currents measured from R334C/E217C-CFTR with addition of the crosslinker MTS-2-MTS in the absence of isoproterenol (ISO), used to activate channels. Login to comment
178 C: Representative trace and summary data for currents measured from R334C/E217C-CFTR with the crosslinker MTS-2-MTS added in the continuing presence of isoproterenol. Login to comment
266 (TIF) Figure S6 Effects of 1 mM MTS2-2MTS on R334C-CFTR and E217C-CFTR channels. Representative traces (left) and summary data (right) for macroscopic currents measured from R334C- (A) and E217C-CFTR (B) by two-electrode voltage clamp. Login to comment
270 MTS-2-MTS also appeared to covalently decrease macroscopic current at R334C-CFTR, but not E217C-CFTR, perhaps due to alteration of charge or side-chain volume. Login to comment
275 (TIF) Figure S7 Effects of MTSET (ET+) and MTSES (ES-) on R334C/E217C-CFTR channels. Representative traces (left) and summary data (right) for macroscopic currents measured from R334C/E217C-CFTR by two-electrode voltage clamp with addition of the 1 mM monofunctional MTS reagents MTSET+ (ET+ ) or MTSES2 (ES2 ) in the presence of isoproterenol (ISO). Login to comment
277 Both ET+ and ES2 covalently bound to R334C/E217C-CFTR. Login to comment
284 (TIF) Figure S8 Distances between residues in R334C-E217C Double Mutant. Login to comment

PMID: 25024266 [PubMed] Cui G et al: "Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR."

No. Sentence Comment

100 AMP-PNP also locks mutant R334C-CFTR into a long stable s2 state (Fuller et al., 2005; Zhang et al., 2005b). Login to comment
109 Therefore, we performed experiments to investigate the modification of WT-, D110C-, E116C-, and R117C-CFTR by MTSET (ET+ ) and MTSES (ES&#e032; ) with the TEVC technique; R334C-CFTR was used as a positive control (Zhang et al., 2005b). Login to comment
113 In contrast, both ET+ and ES&#e032; induced functional covalent modification of R334C-CFTR: ET+ increased macroscopic current by 39% compared with Fig. 2 A. Login to comment
130 This is similar to our previous findings for TM6 mutants R334C-, R352A-, R347C/H-CFTR (Cotten and Welsh, 1999; Zhang et al., 2005b; Cui et al., 2008). Login to comment
189 The data presented so far resolve our first two questions in this paper: (1) Charge-swapping mutations of D110, E116, and R117 of ECL1 destabilize the open state, indicating that these residues contribute to maintaining the outer mouth open pore architecture of CFTR; (2) based Figure 4.ߓ Effects of 1 mM MTSET+ (ET+ ) and MTSES&#e032; (ES&#e032; ) on WT-, D110C-, E116C-, R117C-, and R334C-CFTR. Login to comment
321 This is in contrast to the ability of MTS-2-MTS to lock R352C/D993C-CFTR into the open state or to lock R334C/E217C-CFTR into the closed state (Cui et al., 2013; Rahman et al., 2013). Login to comment

PMID: 25675504 [PubMed] Gao X et al: "Localizing a gate in CFTR."

No. Sentence Comment

78 State-Dependent Reactivity of T338C, F337C, and R334C Implicates the Location of a Gate for CFTR. Login to comment
88 Similar observations were made for F337C-CFTR and R334C-CFTR (Fig. S4 A-D). Login to comment
96 Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment
99 This slight difference in reaction rates between single and double mutants was also seen for R334C, which is one helical turn external to positions 337. Login to comment
100 As shown in Fig. 3A, in the presence of forskolin, 20 bc;M external [Au(CN)2]- readily abolished whole-cell R334C-CFTR currents. Login to comment
102 Similar experiments were performed with the double mutant R334C/G1349D (Fig. 3B). Login to comment
103 Fitting the current decays upon addition of [Au(CN)2]- yielded the reaction rates of 403 &#b1; 20 /M/s (n = 7) and 537 &#b1; 56 /M/s (n = 6) for R334C and R334C/G1349D, respectively. Login to comment
104 Although the Po of R334C-CFTR cannot be assessed due to a greatly reduced single-channel amplitude, by comparing macroscopic current amplitudes in a large number of patches (Fig. 3E), we verified a more than 10-fold decrease of Po by introducing the G1349D mutation. Login to comment
140 (A) External application of 20 bc;M [Au(CN)2]- diminished over 80% of forskolin (Fsk)-activated R334C-CFTR currents. Login to comment
143 (B) [Au(CN)2]- reacted with R334C/G1349D at a faster rate than that with R334C. Login to comment
153 (E) Comparisons of the mean current amplitude between R334C-CFTR and R334C/G1349D-CFTR and between K335C-CFTR and K335C/G1349D-CFTR in excised inside-out patches. Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment

PMID: 26209275 [PubMed] Cui G et al: "Murine and human CFTR exhibit different sensitivities to CFTR potentiators."

No. Sentence Comment

110 The amplitude of s1 was b03;25% and s2 was b03;65% of f, which is different from the ratios of s1 and s2 to f in WT-, R334C-, R352A-, and R347A-hCFTR (s1 is b03;40% and s2 is b03;70% of f) (21, 27, 28). Login to comment
113 As we previously reported, R334C-, R347A-, and R352A-hCFTR generally open from the closed state (c), to s1, then opened to s2 and f states (6, 37, 40). Login to comment
131 WT 0.0 0.1 0.2 0.3 Fractional inhibition by 2.5 &#b5;M GlyH-101 # R334C R334A T338A R352A # # # 0.4 0.5 0.4 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO R334C- hCFTR A B C D ND96 ISO ISO+ GlyH ND96 ISO T338A-hCFTR 1 &#b5;A 50 s 1.0 &#b5;A 50 s ND96 ISO ISO+ GlyH ND96 ISO WT-hCFTR Fig. 5. Login to comment
132 Effects of GlyH-101 on wild-type (WT)- (A), R334C- (B), and T338A-hCFTR (C). Login to comment
180 GlyH-101 at 2.5 òe;M blocked WT-hCFTR 28% at VM afd; afa;60 mV in the continuing presence of 10 òe;M ISO (used to activate CFTR via the beta2-adrenergic receptor; see MATERIALS AND METHODS), but the blocking effect was completely lost with both the R334A and R334C mutations (Fig. 5). Login to comment