ABCMdb

ABCC7 p.Lys335Cys

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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

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
113 For K335C, they reported ~13% decrease in the normalized current (@ -100 mV) and we observed ~30% decrease in the conductance (@Erev). Login to comment
117 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
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
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
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
200 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
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
212 Covalent modification of K335C CFTR altered both conductance and shape of the I-V relation. Login to comment
220 (C) I-V plots for K335C CFTR before and after application of MTS reagents. Login to comment
365 (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

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

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
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
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
145 D, effect of MTSEA on whole cell conductance of K335C-CFTR in 1371E (WT; black), and in 1371Q (gray) backgrounds. Login to comment
151 MTSEA had a small potentiating effect on K335C in the wild type channel background, whereas it was inhibitory in the "locked open" 1371Q channels (Fig. 3D). 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
159 In contrast, I331C, L333C, and K335C reacted faster in the Glu1371 background (Fig. 4, B and C). Login to comment
160 These results reveal clearly that modification of I331C, L333C, and K335C by both these reagents was much slower in the Gln1371 mutational background than in the WT Glu1371 channels. Login to comment
162 The difference in reaction rates between WT and Gln1371 channels was greatest for K335C, which reacted nearly 800 times more slowly in E1371Q background. 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
188 EvidenceforTM6MovementAssociatedwithChannelGating- The state-dependent reactivity of the MTS reagents with I331C, L333C, and K335C channels could indicate a change in the water accessibility of these residues caused by a conformational change in TM6 or by an alteration in the local environment surrounding these residues. Login to comment
195 In contrast, MTSET was without significant effects in both WT and K335C channels on either Po or single channel conductance. 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
198 A, typical recording of whole cell conductance of oocytes expressing K335C-CFTR. 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
232 Therefore, we cannot exclude the possibility that the combination of these two mutations, K335C and E1371Q is responsible for the observed changes in reactivity of K335C. Login to comment
258 The openprobabilityandmeanburstandinterbursttimes(meansϮS.E.)forI331C,L333C,K335C,andWTchannels in untreated (-MTSET) and MTSET-treated (ϩMTSET) conditions are shown. 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

74 In fact, similar charge-dependent effects were observed in R334C, K335C, T338C, and S341C (Fig. 3). 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
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
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

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
107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment

PMID: 9922375 [PubMed] Sheppard DN et al: "Structure and function of the CFTR chloride channel."

No. Sentence Comment

112 On mutants K95C and K335C interact with methanethiosulfo- the basis of these data, the minimum diameter of the nate (MTS) reagents, and mutations that eliminate the CFTR pore was estimated to be Ç5.3 A˚ (77), similar to positive charge at K335 reduce single-channel conduc- that reported for other Cl0 channels (10, 20, 55). 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

146 The rate of modification of L333C and K335C, also at the extracellular end of TM6, was also decreased in an E1371Q background, suggesting slower modification of open, compared with closed channels (26). 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
152 L102C, like T338C, becomes apparently more accessible to internal cysteine reactive reagents in open channels (Fig. 6B), but is inaccessible to extracellular MTSES (Fig. FIGURE 4. Login to comment
153 Rate of modification of T338C by external MTSES. Login to comment

PMID: 9511930 [PubMed] Akabas MH et al: "Probing the structural and functional domains of the CFTR chloride channel."

No. Sentence Comment

81 The effect of modification of the K335C mutant by the MTS reagents on CFTR current would be indirect. 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
162 In this case the effects of modification on the K335C mutant by the MTS reagents would have to be indirect. 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
160 In this case the effects of modification on the K335C mutant by the MTS reagents would have to be indirect. Login to comment

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

No. Sentence Comment

89 For reasons unclear at this juncture, application of 1 mM [Au(CN)2]- to inside-out patches containing K335C-CFTR only resulted in reversible block, although this residue has been previously implicated as pore-lining (Fig. S4E) (21). Login to comment
90 In addition, 1 mM [Au(CN)2]- applied in the absence of ATP also hardly altered the K335C-CFTR current in inside-out patches (Fig. S4F). 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
107 When 1 mM [Au(CN)2]- was applied to the external side of the cell expressing K335C-CFTR mutants, the whole-cell current decreased in two steps (Fig. 3C). Login to comment
109 These results, together with the only blocking effect of 1 mM [Au(CN)2]- being seen on K335C-CFTR in inside-out patches in the presence of ATP (Fig. S4E), indicate that the side chain of 335C is not well exposed in the open state. Login to comment
110 However, after G1349D was introduced into K335C-CFTR to lower its Po, 50 bc;M [Au(CN)2]- could readily react with 335C with a reaction rate of 1,809 &#b1; 201 /M/s (n = 5) (Fig. 3D). Login to comment
146 (C) Reaction of K335C-CFTR channels with [Au(CN)2]- . Login to comment
148 The addition of 1 mM [Au(CN)2]- led to a biphasic decay of forskolin-activated K335C-CFTR currents: a faster phase of blockade and a slower phase of ligand exchange reaction. Login to comment
149 (D) Reaction of K335C/G1349D-CFTR with [Au(CN)2]- . 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