ABCMdb

PMID: 11585852

Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC
CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.
J Gen Physiol. 2001 Oct;118(4):407-31., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Arg334Cys Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. Login to comment 25 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys R347C and R352C were gifts of M. Akabas (Albert Einstein College of Medicine, Bronx, NY) and used in their parent vector, pMN. Login to comment 60 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg347Cys ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys 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 113 ABCC7 p.Lys335Cys For K335C, they reported ~13% decrease in the normalized current (@ -100 mV) and we observed ~30% decrease in the conductance (@Erev). Login to comment 115 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 117 ABCC7 p.Lys335Cys For K335C CFTR, Cheung and Akabas (1996) reported that the application of 2.5 mM MTSEA resulted in ~20% decrease in the normalized current (@ -100 mV), whereas we observed ~20% increase in the conductance (@Erev). Login to comment 122 ABCC7 p.Arg347Cys Shown in Fig. 4 is a representative experiment in which an oocyte expressing R347C CFTR was exposed to MTS reagents. Login to comment 124 ABCC7 p.Arg347Cys The experiments were repeated with each of the compounds individually, both with and without prior exposure to 2-ME and in no case were we able to detect an effect on the function of R347C CFTR. Login to comment 125 ABCC7 p.Arg347Cys Cheung and Akabas (1996) initially reported that there was a ‫%05ف‬ decrease in the normalized current (@ -100 mV) after exposure of oocytes expressing R347C CFTR to 10 mM MTSES, 1 mM MTSET or 2.5 mM MTSEA. Login to comment 126 ABCC7 p.Arg347Cys However, in the course of subsequent studies by Cheung and Akabas (1997), it was discovered that the R347C construct had a substantial deletion. Login to comment 127 ABCC7 p.Arg347Cys MTSES or MTSET were reported as not reacting with the full-length R347C CFTR, and MTSEA was reported to cause a 19% inhibition. Login to comment 129 ABCC7 p.Arg352Cys In the case of R352C CFTR, our observations also differed substantially from those reported by Cheung and Akabas (1996, 1997). Login to comment 131 ABCC7 p.Arg352Cys In contrast, we were not able to detect effects of this magnitude applying either MTSES or MTSET to oocytes expressing R352C CFTR (Fig. 5 A). Login to comment 133 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys Comparison of the effects of MTSES, MTSET, and MTSEA on the conductance of oocytes expressing R334C, K335C, R347C, or R352C CFTR. Login to comment 137 ABCC7 p.Arg347Cys MTS reagents did not discernibly alter the function of R347C CFTR expressed in oocytes. Login to comment 140 ABCC7 p.Arg352Cys MTSET and MTSES, polar thiol reagents, did not produce a discernible alteration in conductance of oocytes expressing R352C CFTR. Login to comment 144 ABCC7 p.Arg352Cys (B) Addition of 1 mM MTSEA produced a sixfold increase in conductance of an oocyte expressing R352C CFTR not previously exposed to MTSET. Login to comment 146 ABCC7 p.Arg352Gln (C) MTSEA produced a twofold increase in conductance in an oocyte expressing R352Q CFTR, which spontaneously reversed when MTSEA was removed from the perfusate. Login to comment 148 ABCC7 p.Arg352Gln ABCC7 p.Arg352His Changes of this magnitude are also seen in oocytes expressing R352Q or R352H CFTR, but were not seen in oocytes expressing wt CFTR. Login to comment 151 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys R352C CFTR Function Is Modified by Addition of 1 mM MTSEA to the Perfusate, However the Effect Is Not Cysteine-specific Shown in Fig. 5 B is the result of a representative exper- iment in which an oocyte expressing R352C CFTR was exposed to 1 mM MTSEA; the result being a sixfold increase in conductance. Login to comment 153 ABCC7 p.Arg334Cys 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 154 ABCC7 p.Arg352Gln ABCC7 p.Arg352His Importantly, we also obtained a transient response to the application of MTSEA in oocytes expressing R352Q CFTR (Fig. 5 C) or R352H CFTR (unpublished data). Login to comment 157 ABCC7 p.Arg352Gln Guinamard and Akabas (1999) reported that substitutions for R352 (C, Q, and H) decreased the Cl-/Naϩ selectivity of CFTR as determined in detached patches (CHO cells), but we did not detect any alteration in the reversal potential associated with the expression of R352Q or H CFTR in oocytes. Login to comment 159 ABCC7 p.Arg334Cys Covalent modification of R334C CFTR was stable, reproducible, and varied with the electrostatic nature of the moiety covalently attached. Login to comment 166 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys R334C CFTR did not exhibit time-dependent current relaxations. Login to comment 178 ABCC7 p.Arg334Cys (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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys (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 186 ABCC7 p.Arg352Cys In view of the results obtained with R352C CFTR, we tested for effects of exposure to MTSET and MTSES using constructs bearing substitutions other than cysteine for R334. Login to comment 187 ABCC7 p.Arg334Gln ABCC7 p.Arg334Ala MTSES, MTSET, or MTSEA (100 ␮M or 1 mM) were added to the perfusate of oocytes expressing R334A or R334Q CFTR and produced no discernible effect on conductance (unpublished data). Login to comment 188 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys 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 200 ABCC7 p.Lys335Cys Fig. 8 A contains the results of a representative experiment in which the total membrane conductance (@ Erev) of an oocyte expressing K335C CFTR was measured as a function of time. After achieving steady-state activation, 2-ME (1 mM) was added to the bath and produced a slight increase in conductance. Login to comment 207 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys 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 212 ABCC7 p.Lys335Cys Covalent modification of K335C CFTR altered both conductance and shape of the I-V relation. Login to comment 220 ABCC7 p.Lys335Cys (C) I-V plots for K335C CFTR before and after application of MTS reagents. Login to comment 225 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys (A) Conductance of oocytes expressing R334C CFTR determined just after modification by MTSET plotted versus premodification conductance. Login to comment 244 ABCC7 p.Arg334Cys Dashed lines are mean values of RR for modified and unmodified R334C CFTR. Login to comment 246 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys Covalent modification of R334C increased single-channel conductance. Login to comment 257 ABCC7 p.Arg334Cys (A) Representative traces from an oocyte expressing R334C CFTR in the detached, inside-out patch configuration, Vm ϭ -80 mV. Login to comment 266 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys Covalent modification of a single R334C CFTR channel in real time by including MTSET in the patch pipette. Login to comment 283 ABCC7 p.Arg334Cys (C) Po determined before and after covalent modification for four, single R334C CFTR channels. Login to comment 287 ABCC7 p.Arg334Cys pH titration of oocytes expressing R334C CFTR altered both the conductance and the shape of the I-V relation. Login to comment 288 ABCC7 p.Arg334Cys (A) I-V plot from an oocyte expressing R334C CFTR. Login to comment 293 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys Functional effect of covalent modification of R334C CFTR was dependent on the bath pH. Login to comment 310 ABCC7 p.Arg334Cys Shown in Fig. 14 are the results of modifying R334C CFTR with two "neutral" reagents, NEM and MTSHE. Login to comment 313 ABCC7 p.Arg334Gln The conductance of oocytes expressing R334Q CFTR was independent of pH and was not greatly altered by NEM. Login to comment 314 ABCC7 p.Arg334Cys 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 317 ABCC7 p.Arg334Gln Exposure of oocytes expressing R334Q CFTR to MTSHE produced only small changes in conductance. Login to comment 318 ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334His 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 320 ABCC7 p.Arg334His An I-V plot from a representative experiment in which the pH of the solution bathing an oocyte expressing R334H CFTR was acidified from 7.4 to 6.02 and then 4.80 is shown in Fig. 15 A. Login to comment 324 ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys Modification of R334C CFTR with neutral reagents caused pH-dependent changes in conductance. Login to comment 331 ABCC7 p.Arg334Cys (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 341 ABCC7 p.Lys335Ala ABCC7 p.Arg334His However, for two mutants, R334H and K335A, the RR value clearly deviated significantly from that predicted by the general trend. Login to comment 344 ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys 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 350 ABCC7 p.Arg334His pH titration of oocytes expressing R334H CFTR altered both the conductance and the shape of the I-V relation. Login to comment 351 ABCC7 p.Arg334His (A) I-V plots from an oocyte expressing R334H CFTR. Login to comment 364 ABCC7 p.Arg334Cys (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 365 ABCC7 p.Lys335Cys (B) RR is plotted as a function of the net charge of the amino acid at position 335 where glutamic acid (E) and aspartic acid (D) were assigned a value of -1, alanine (A) was treated as neutral, cysteine (C) was also treated as neutral because K335C CFTR exhibited a smaller pH responses and its apparent pKa was not determined, and lysine (K) and arginine (R) were assigned a value of ϩ1. Login to comment 372 ABCC7 p.Arg334Cys The effects of covalent modification of R334C CFTR could be simulated using charged vestibule models (see appendix). Login to comment 373 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys (closed circles) Unmodified R334C CFTR, ⌿o ϭ 0. Login to comment 378 ABCC7 p.Arg334Cys (open circles) MTSET-modified R334C CFTR, ⌿o ϭ ϩ 50 mV. Login to comment 379 ABCC7 p.Arg334Cys (closed triangles) MTSES-modified R334C CFTR, ⌿o ϭ -10 mV. Login to comment 381 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys (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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334His 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 ABCC7 p.Arg334Cys 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 435 ABCC7 p.Arg352Cys Even if we allow for the possibility of some modest accessibility of R352C, it is evident that the outer sites are highly reactive, whereas the inner sites are either not at all reactive or much less so. Login to comment 442 ABCC7 p.Arg352Cys This is an important point because in the course of our experiments (e.g., R352C CFTR) and those of others using cysteine accessibility or pH titration (Coulter et al., 1995; Akabas, 1998), it has become apparent that structural changes can render sites "distant" from the mutation accessible or titratable. Login to comment 443 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Trp ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys 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 483 ABCC7 p.Tyr380Cys The conductance of oocytes expressing the I379C construct was reduced by MTS reagents (MTSET, MTSEA, and MTSES) regardless of charge, whereas the effects of the same reagents on oocytes expressing the Y380C construct were distinctly charge-dependent. Login to comment 484 ABCC7 p.Tyr380Cys The anionic reagent (MTSES) increased the macroscopic conductance of oocytes expressing Y380C and altered the shape of the I-V plot from outwardly rectifying to nearly linear, whereas cationic reagents (MTSET and MTSEA) reduced the conductance and enhanced the rectification, exactly as predicted for a residue that resides near the mouth of a cation-selective pore. Login to comment 485 ABCC7 p.Tyr380Cys Single-channel records disclosed that MTS reagents altered the unitary conductance of Y380C channels as would be predicted from macroscopic records, but did not affect channel gating. Login to comment