ABCMdb

PMID: 18056267

Beck EJ, Yang Y, Yaemsiri S, Raghuram V
Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3., 2008-02-22 [PubMed]

Sentences

No. Mutations Sentence Comment

85 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 94 ABCC7 p.Lys329Cys Cd2ϩ had a small but significant potentiating effect on K329C channels, but MTSEA, which by itself is without any significant effect, was able to abolish the potentiating effect of Cd2ϩ (supplemental Fig. S2). Login to comment 100 ABCC7 p.Arg347Cys ABCC7 p.Gln353Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr351Cys ABCC7 p.Met348Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Lys329Cys ABCC7 p.Phe342Cys ABCC7 p.Val350Cys ABCC7 p.Gly330Cys ABCC7 p.Tyr325Cys ABCC7 p.Leu327Cys ABCC7 p.Ser341Cys ABCC7 p.Val345Cys ABCC7 p.Ile332Cys ABCC7 p.Ile336Cys ABCC7 p.Ile340Cys ABCC7 p.Ala349Cys ABCC7 p.Ala326Cys ABCC7 p.Ile328Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys ABCC7 p.Thr339Cys 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 ABCC7 p.Arg334Cys 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 ABCC7 p.Arg334Cys An example of this protocol applied to R334C-CFTR expressing oocytes is shown in Fig. 2A. Login to comment 117 ABCC7 p.Arg334Cys 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 120 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln Functional Effects of Cd2ϩ and MTSEA on Substituted Cysteines in E1371Q-CFTR-To study the accessibility of substituted cysteines in the open state, the effects of Cd2ϩ and MTSEA were examined for CFTR channels bearing the E1371Q mutation. Login to comment 123 ABCC7 p.Glu1371Gln Hence, the observed effects of Cd2ϩ and MTS reagents, on E1371Q CFTR represent effects on open channel state. Login to comment 124 ABCC7 p.Glu1371Gln As shown in Fig. 3A, the open probability (Po) of wild type CFTR channels was about 0.2, whereas it was nearly 1 for E1371Q-CFTR. Login to comment 127 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 ABCC7 p.Arg334Cys A, representative experiment in which MTSEA (1 mM) was applied for 3 min to R334C-CFTR channels that are closed. Login to comment 131 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys B, summary of effects of Cd2ϩ on MTSEA-modified I331C, L333C, R334C, K335C, and T338C channels. Login to comment 135 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Ile331Cys 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 137 ABCC7 p.Glu1371Gln A, effect of E1371Q mutation on CFTR channel activity. Login to comment 138 ABCC7 p.Glu1371Gln Representative single-channel current traces of WT (1371E; upper trace), and E1371Q (lower trace) CFTR. Login to comment 140 ABCC7 p.Glu1371Gln Note the time scale for E1371Q record. Login to comment 141 ABCC7 p.Glu1371Gln The mean Po of WT and E1371Q CFTR was 0.28 Ϯ 0.02 (n ϭ 7) and 0.93 Ϯ 0.02 (n ϭ 7), respectively. Login to comment 142 ABCC7 p.Leu333Cys B, effect of Cd2ϩ on whole cell conductance of L333C-CFTR in 1371E (WT; blue) and in 1371Q (red) backgrounds. Login to comment 145 ABCC7 p.Lys335Cys D, effect of MTSEA on whole cell conductance of K335C-CFTR in 1371E (WT; black), and in 1371Q (gray) backgrounds. Login to comment 151 ABCC7 p.Lys335Cys 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 152 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 154 ABCC7 p.Glu1371Gln The differences in the magnitude of MTS modification suggest that in E1371Q background there is a significant change in TM6 structure. Login to comment 156 ABCC7 p.Glu1371Gln Effect of E1371Q Mutation on Thiol Modification Rates-To obtain quantitative information about changes in reactivity, the rates of substituted cysteine modification were calculated by fitting the time course of Cl-conductance modification with a single exponential, which was then used to calculate second order reaction rate constants for various MTS reagents. Login to comment 158 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys 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 159 ABCC7 p.Lys335Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys In contrast, I331C, L333C, and K335C reacted faster in the Glu1371 background (Fig. 4, B and C). Login to comment 160 ABCC7 p.Lys335Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 ABCC7 p.Lys335Cys ABCC7 p.Glu1371Gln 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 163 ABCC7 p.Leu333Cys L333C showed a relatively smaller 10-fold 100 75 50 4803602401200 s 1371E; 100 µM 1371Q; 100 µM 100 90 80 70 240180120600 s 1371E; 1 mM 1371Q; 1 mM 120 100 80 120600 s 1371E; 10 µM 1371Q; 1 mM 100 75 50 25 180120600 s 1371Q; 10 µM 1371E; 10 µM 100 90 80 180120600 s 1371Q; 1 mM 1371E; 1 mM 100 75 50 4803602401200 s 1371E; 100 µM 1371Q; 100 µM 9060300 s 1371E; 10 µM 1371Q; 1 mM 180120600 s 1371E; 10 µM 1371Q; 10 µM 100 75 50 25 180120600 s 1371E; 10 µM 1371Q; 10 µM 140 130 120 110 100 4530150 s 1371Q; 10 µM 1371E; 10 µM FIGURE 4. Login to comment 164 ABCC7 p.Glu1371Gln MTSEA, and MTSES modification rates of residues in TM6 in WT and E1371Q backgrounds. Login to comment 168 ABCC7 p.Glu1371Gln ABCC7 p.Ile331Cys CFTR Conformation Changes during Gating FEBRUARY 22, 2008•VOLUME 283•NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 4961 decrease, and I331C reacted 5-fold slower (Fig. 4, B and C) in E1371Q channels. Login to comment 172 ABCC7 p.Glu1371Gln Alternatively, it is possible that the observed reaction rates in E1371Q mutational background are due to a change in the channel structure induced by the mutation and not related to structural changes associated with open channel state. Login to comment 178 ABCC7 p.Leu333Cys All of the residues except L333C had varying but significant differences in the magnitude of the functional effect by MTS reagents. Login to comment 179 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys 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 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys MTSES, however, had a smaller inhibitory effect on R334C and K335C when minimally activated. Login to comment 181 ABCC7 p.Ile331Cys For I331C, the inhibitory effect of both MTSEA and MTSES was larger under minimally active conditions. Login to comment 184 ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys 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 185 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys However, I331C and L333C channels had a significantly faster modification rate, when minimally active. Login to comment 186 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys When stimulated by 0.02 mM IBMX, both I331C and L333C reacted nearly 25 times faster with MTSEA and nearly 10-20 times faster with MTSES. Login to comment 187 ABCC7 p.Ile331Cys These results suggest that when CFTR Channel Po is low, residues I331C and L33C react quite rapidly with MTS reagents, and as the Po increases their reactivity decreases correspondingly. Login to comment 188 ABCC7 p.Lys335Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 192 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys For two of the three mutants, I331C and L333C, modification with MTSET profoundly affected channel gating (Fig. 7A). Login to comment 193 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys The open probabilities of MTSET-modified I331C and L333C channels were significantly smaller than those of unmodified channels. Login to comment 195 ABCC7 p.Lys335Cys In contrast, MTSET was without significant effects in both WT and K335C channels on either Po or single channel conductance. Login to comment 196 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys These results indicate that MTSET reduces the whole cell conductance of I331C- and L333C-expressing oocytes by inhibiting channel gating and not by affecting channel permeation properties. Login to comment 197 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Ile331Cys ABCC7 p.Ile331Cys ABCC7 p.Ile331Cys 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 ABCC7 p.Lys335Cys A, typical recording of whole cell conductance of oocytes expressing K335C-CFTR. Login to comment 213 ABCC7 p.Lys329Cys We believe that K329C, whose whole cell conductance was stimulated by Cd2ϩ , is an example of one such residue that reacts with MTSEA, but the modification is without effect on channel function (supplemental Fig. S2). Login to comment 216 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 217 ABCC7 p.Thr338Cys That study did not observe any effects of MTSEA on T338C channels, whereas we did here. Login to comment 218 ABCC7 p.Arg347Cys ABCC7 p.Arg352Cys ABCC7 p.Gln353Cys ABCC7 p.Thr351Cys ABCC7 p.Ser341Cys ABCC7 p.Ile344Cys ABCC7 p.Phe337Cys Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment 220 ABCC7 p.Arg347Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys 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 222 ABCC7 p.Glu1371Gln State-dependent Reactivity Reflects a Local Rearrangement and Exposure of the Side Chains-We observed large differences in the rates of cysteine modification at three (residues 331, 333, and 335) of the five residues in the E1371Q background. Login to comment 223 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys ABCC7 p.Ile331Cys 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 ABCC7 p.Lys335Cys ABCC7 p.Lys335Cys ABCC7 p.Glu1371Gln 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 239 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys 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 240 ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys It must be pointed out that under low IBMX concentrations, a 5-fold decrease in CFTR Po cannot account for the entire differences in reactivity of I331C and L333C. Login to comment 242 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Leu333Cys ABCC7 p.Ile331Cys 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 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys 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 246 ABCC7 p.Lys1250Ala The authors suggested that the slower reactivity in the open state that is stabilized in the K1250A mutant channel was due to changes in the local electrostatic environment (see above). Login to comment 247 ABCC7 p.Arg334Cys ABCC7 p.Glu1371Gln 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 249 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Gln A notable difference between the two mutations is that the Walker A mutation K1250A, unlike E1371Q, decreases the ATP binding affinity of NBD2 (27), which profoundly reduces the channel opening rate (28, 29) in addition to decreasing the closing rate (30) of CFTR. Login to comment 250 ABCC7 p.Lys1250Ala Hence, the slower modification rate observed in the earlier study (26) may be specific to K1250A and not a general characteristic of the open state. Login to comment 258 ABCC7 p.Lys335Cys ABCC7 p.Leu333Cys The openprobabilityandmeanburstandinterbursttimes(meansϮS.E.)forI331C,L333C,K335C,andWTchannels in untreated (-MTSET) and MTSET-treated (ϩMTSET) conditions are shown. Login to comment