ABCMdb

PMID: 25944907

El Hiani Y, Linsdell P
Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.
J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5., [PubMed]

Sentences

No. Mutations Sentence Comment

45 ABCC7 p.Arg303Cys Altering the side chain charge at this position (either by mutagenesis or by covalent modification of R303C by charged cysteine-reactive methanethiosulfonate (MTS) reagents) suggested that the positive charge at this position played a key role in attracting Clafa; ions electrostatically from the cytoplasm to the inner mouth of the pore (19). Login to comment 51 ABCC7 p.Val510Ala In this study, we used a human CFTR variant in which all cysteines had been removed by mutagenesis (as described in Ref. 21) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (22). Login to comment 103 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys A-D, examples of modification of four different mutants (A, R303C, used in this study as a positive control; B, K190C; C, K370C; D, R1048C) by MTSES (left panels) or MTSET (right panels). Login to comment 109 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Lys946Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg251Cys ABCC7 p.Arg975Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys Application of MTSES (200 òe;M) following channel activation with PKA and ATP never caused an increase in macroscopic current amplitude but decreased current amplitude in K190C, R248C, R251C, R303C, K370C, K946C, R975C, K1041C, and R1048C (Figs. 2 and 3). Login to comment 110 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Lys946Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg251Cys ABCC7 p.Arg975Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys The effect of MTSET on these MTSES-sensitive mutants was to increase (K190C and R303C), decrease (R248C, K946C, K1041C, and R1048C), or have no effect (R251C, K370C, and R975C) on macroscopic current amplitude (Figs. 2 and 3). Login to comment 111 ABCC7 p.Arg1162Cys ABCC7 p.Lys254Cys ABCC7 p.Arg297Cys ABCC7 p.Lys298Cys ABCC7 p.Lys294Cys ABCC7 p.Lys1165Cys ABCC7 p.His1085Cys Other mutants studied (K254C, K294C, R297C, K298C, H1085C, R1162C, and K1165C) were not significantly affected by either MTSES or MTSET (Fig. 3), suggesting that cysteine side chains at these positions were not modified by cytoplasmic MTS reagents. Login to comment 112 ABCC7 p.Lys978Cys ABCC7 p.Arg153Cys ABCC7 p.His949Cys We did not observe any ATPand PKA-dependent, CFTRinh-172-sensitive macroscopic currents in inside-out patches associated with R153C, H949C, or K978C (Fig. 3), most likely due to lack of channel expression in the membrane (11). Login to comment 114 ABCC7 p.Arg303Cys ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys The effects of MTS modification of side chains within the ICLs could reflect changes in Clafa; conductance (as suggested previously for R303C (19)) or in open probability (as shown previously for K946C and R975C (11)) or a combination of the two. Login to comment 116 ABCC7 p.Arg303Cys ABCC7 p.Arg303Cys ABCC7 p.Lys946Cys ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys ABCC7 p.Arg975Cys Results with R303C, K946C, and R975C, which as described above are expected to affect predominantly Clafa; conductance (R303C) or gating (K946C and R975C), suggest that use of PPi in this way can effectively separate effects on Clafa; conductance from those on gating. Login to comment 117 ABCC7 p.Arg303Cys Thus, the effects of MTSES and MTSET on R303C were preserved following PPi treatment, consistent with a gating-independent effect on Clafa; conductance (Figs. 4 and 5). Login to comment 118 ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys In contrast, the effects of MTS reagents on both K946C and R975C were lost following PPi treatment (Fig. 5), consistent with these reagents modifying the normal gating process (reducing channel open probability) without affecting Clafa; conductance (11). Login to comment 119 ABCC7 p.Arg251Cys The effects of MTS reagents on R251C were similarly lost following PPi treatment, suggesting that this mutant also had little or no effect on Clafa; conductance (Fig. 5). Login to comment 120 ABCC7 p.Lys1041Cys Inhibitory effects of MTS reagents on K1041C were diminished but not completely abolished following PPi treatment (Fig. 5). Login to comment 121 ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys In contrast, the inhibitory effects of MTSES on K190C, R248C, K370C, and R1048C were preserved following PPi treatment (Figs. 4 and 5), suggesting that modification with this negatively charged reagent caused a major inhibition of Clafa; conductance in open channels in each of these mutants. Login to comment 122 ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys Interestingly, in each of these four mutants, MTSET caused a significant increase in macroscopic current amplitude when applied after PPi treatment (Figs. 4 and 5) even though MTSET applied in the absence of PPi treatment decreased macroscopic current amplitude in R248C, K370C, and R1048C (Figs. 4 and 5). Login to comment 123 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys This implies that modification with positively charged MTSET caused an increase in Clafa; conductance in each of K190C, R248C, R303C, K370C, and R1048C, an effect that may potentially be masked by inhibitory effects on channel open probability in normally gating channels. Login to comment 126 ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys As described previously (11) and consistent with results from macroscopic current recording following channel treatment with PPi (Fig. 5), application of MTSES or MTSET had no effect on current amplitude in K946C or R975C (Figs. 6 and 7). Login to comment 127 ABCC7 p.Arg251Cys Current amplitude in R251C was slightly increased by MTSET but not significantly altered by MTSES (Figs. 6 and 7). Login to comment 132 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys Error bars represent the means afe; S.E. from three to five patches. Cytoplasmic Entrance to the CFTR Channel Pore JUNE 19, 2015ߦVOLUME 290ߦNUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 15859 rent amplitude in each of K190C, R248C, R303C, K370C, K1041C, and R1048C. Login to comment 139 ABCC7 p.Lys190Cys Following channel activation with PKA and ATP, channels were "locked" in the open statebyapplicationofPPi (2mM;asindicatedbyhatchedbars)priortoadditionofMTSreagent(200òe;M;asindicatedbywhitebars).TheidentityofCFTRcurrents was confirmed by sensitivity to CFTRinh-172 (-172; 5 òe;M; indicated by black bars) at the end of the recording except for very small residual currents for K190C following MTSES modification. Login to comment 141 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Lys946Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg251Cys ABCC7 p.Arg975Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys Cytoplasmic Entrance to the CFTR Channel Pore 15860 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 25ߦJUNE 19, 2015 at SEMMELWEIS UNIV OF MEDICINE on December 4, amplitudes in unmodified channels was Cys-less b; K946C b03; R975C b0e; K370C b0e; R251C b0e; K1041C b03; R248C b0e; R1048C b0e; R303C b0e; K190C (Fig. 7A). Login to comment 142 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys Among channels showing MTSES-sensitive current amplitudes, the order following modification was K1041C b0e; R1048C b0e; K370C b0e; R248C b0e; R303C b0e; K190C (Fig. 6, B and C, and 7B). Login to comment 155 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys ABCC7 p.Lys1041Cys Single channel recording indicated directly that for each of K190C, R248C, R303C, K370C, K1041C, and R1048C Clafa; conductance was reduced relative to Cys-less (Figs. 6 and 7A), Clafa; conductance was further reduced by modification by negatively charged MTSES (Figs. 6 and 7B), and Clafa; conductance was restored to near wild-type (Cys-less) values by modification by positively charged MTSET (Figs. 6 and 7C). Login to comment 164 ABCC7 p.Lys946Cys ABCC7 p.Arg251Cys ABCC7 p.Arg975Cys Consistent with this, the functional effect of MTS reagents on R251C, K946C, and R975C were abolished in channels that had been treated with PPi to maintain the channels in the open state (Fig. 5). Login to comment 165 ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys This suggests that MTS modification of these mutants may have affected channel gating, leading to a change in overall macroscopic current amplitude (Fig. 3), as previously demonstrated directly for K946C and R975C using single channel recording (11). Login to comment 166 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Arg1048Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys In contrast, the effects of MTS modification of PPi-treated channels in K190C, R248C, R303C, K370C, and R1048C (Fig. 5) closely mirrored the charge-dependent effects of MTS modification on single channel current amplitude (Figs. 6 and 7). Login to comment 167 ABCC7 p.Lys1041Cys Only in K1041C was there a discrepancy between MTS effects on single channel current amplitude and on macroscopic current amplitude following PPi treatment. Login to comment 170 ABCC7 p.Lys946Cys ABCC7 p.Arg975Cys Effects of both mutagenesis (12, 29-32) and MTS modification (including of K946C and R975C) (11) within the ICLs on channel gating have been reported previously; however, the mechanism(s) by which these manipulations of ICL structure affect channel gating is not well understood. Login to comment 173 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys We believe that the functional importance of Lys-190, Arg-248, Arg-303, and Lys-370 is likely greater than that of Lys-1041 and Arg-1048 because of the greater reduction in single channel current amplitude following introduction of a negative charge by MTSES modification in K190C, R248C, R303C, and K370C FIGURE 6. Login to comment 180 ABCC7 p.Arg1048Cys ABCC7 p.Lys1041Cys Error bars represent the means afe; S.E. from 3-10 patches. Cytoplasmic Entrance to the CFTR Channel Pore 15862 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290ߦNUMBER 25ߦJUNE 19, 2015 at SEMMELWEIS UNIV OF MEDICINE on December , when compared with K1041C and R1048C (Figs. 6C and 7B). Login to comment 181 ABCC7 p.Arg303Cys ABCC7 p.Lys190Cys ABCC7 p.Lys370Cys ABCC7 p.Arg248Cys Single channel current amplitude was very low in MTSES- modified K190C, R248C, R303C, and K370C channels (b0d;25% of Cys-less current amplitude; Figs. 6, B and C, and 7B), suggesting that most permeating Clafa; ions must pass close to these residues. Login to comment 182 ABCC7 p.Arg1048Cys ABCC7 p.Lys1041Cys Modification by MTSES had a lesser effect on unitary current amplitude in K1041C (b03;59% of Cys-less) and R1048C (b03;38% of Cys-less) (Fig. 6, B and C, and 7B) perhaps because these positive charges are located further from the major Clafa; permeation pathway. Login to comment