ABCMdb

PMID: 23955087

Wang W, El Hiani Y, Rubaiy HN, Linsdell P
Relative contribution of different transmembrane segments to the CFTR chloride channel pore.
Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. Login to comment 31 ABCC7 p.Thr338Ala Overall, the effects of the T338A mutation have been interpreted as reflecting an increase in functional diameter of the narrowest part of the pore [26] and a reduced barrier to the movement of permeant ions inside the pore [11]. Login to comment 38 ABCC7 p.Val510Ala In this study, we have used two different human CFTR variants as backgrounds for further mutagenesis: wild type CFTR and "cys-less" CFTR in which all cysteines have been removed by mutagenesis [30] and that also includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane [20]. Login to comment 46 ABCC7 p.Glu1371Gln In some cases, cysteine mutants were combined with the NBD2 mutation E1371Q, which we have used previously to abolish ATP-dependent channel gating and so isolate effects of cysteine reactive substances on open CFTR channels [41-43]. Login to comment 70 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala Because the properties of T338A-CFTR channels have been investigated in detail previously, simplified protocols were used to facilitate comparison of the effects of the T338A mutation with those of other mutations (Figs. 5, 6, 7, and 8). Login to comment 77 ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys ABCC7 p.Ile1112Cys Note that while MTSES caused rapid inhibition of macroscopic current amplitude in T1115C and S1118C, inhibition of I1112C current was much slower (note different time scale for this mutant). Login to comment 78 ABCC7 p.Thr1121Cys MTSES did not affect T1121C currents; however, these were inhibited by CFTRinh-172 (5 bc;M; hatched bar). Login to comment 82 ABCC7 p.Ile1119Cys ABCC7 p.Leu1120Cys Note that TM11 mutants I1119C and L1120C were not tested since they have previously been shown to be unaffected by extracellular MTS reagents and were therefore described as being non-pore lining [10]. Login to comment 83 ABCC7 p.Glu873Cys TM7 mutant E873C expression did not yield functional CFTR channel currents in BHK cell patches approximated from: Kd &#bc; Au CN &#f0; &#de;2 - . Login to comment 87 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala T338A, as well as other substitutions at this position, cause complex changes in the relative permeability of different anions in CFTR [26]; however, the major changes observed in T338A can be summarized as (a) an increase in the relative permeability of lyotropic anions such as SCN- , NO3 - , Br- , I- , and ClO4 - , and (b) an increase in the relative permeability of extracellular organic (kosmotropic) monvalent anions including formate, acetate, propanoate, and pyruvate [26]. Login to comment 104 ABCC7 p.Ile1112Cys Black bars represent a significant difference from cys-less (P<0.05); gray bars (I1112C only) no significant difference. Mean of data from three to six patches Results Accessibility of cysteine side chains introduced into TMs 5, 7, and 11 We hypothesized that the central portion of the CFTR pore might be lined by TMs 1, 5, 6, 7, 11, and 12 (Fig. 1). Login to comment 107 ABCC7 p.Glu1371Gln Figure 2a shows the effect of internal MTSES on currents carried by some TM11 mutant channels in inside- Fig. 4 Effect of the E1371Q mutation on modification by internal and external MTSES. Login to comment 111 ABCC7 p.Glu1371Gln ABCC7 p.Glu1371Gln ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys b Example whole cell currents recorded continuously at +30 mV for constitutively active T1115C/ E1371Q and S1118C/E1371Q channels. Login to comment 112 ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys In contrast to currents carried by T1115C and S1118C channels (see Fig. 3), these whole cell currents were not significantly affected by addition of 200 bc;M MTSES to the extracellular solution (black bars), even though these currents were positively identified as being carried by CFTR by sensitivity to GlyH-101 (50 bc;M, hatched bars). Login to comment 115 ABCC7 p.Glu1371Gln In both c and d, black bars represent the named mutants in a cys-less background, and white bars in a cys-less/E1371Q background, and asterisks indicate a significant difference between these two backgrounds (P<0.05). Login to comment 117 ABCC7 p.Ser1118Cys ABCC7 p.Thr1121Cys ABCC7 p.Thr1115Cys ABCC7 p.Ile1112Cys ABCC7 p.Phe1116Cys ABCC7 p.Ala1113Cys ABCC7 p.Phe1110Cys ABCC7 p.Phe1111Cys ABCC7 p.Val1114Cys ABCC7 p.Ile1117Cys ABCC7 p.Ile1109Cys Application of MTSES (200 bc;M) following channel activation with PKA and ATP caused a decrease in macroscopic current amplitude in I1112C, T1115C and S1118C, but not in T1121C (Fig. 2a, b) or in I1109C, F1110C, F1111C, A1113C, V1114C, F1116C, or I1117C (Fig. 2c). Login to comment 120 ABCC7 p.Glu873Cys We did not observe any CFTR current in inside-out patches associated with E873C (TM7). Login to comment 122 ABCC7 p.Ser1118Cys ABCC7 p.Thr1121Cys ABCC7 p.Thr1122Cys ABCC7 p.Ile1119Cys ABCC7 p.Leu1120Cys ABCC7 p.Phe1116Cys ABCC7 p.Ile1117Cys Previously it was shown that S1118C, T1121C, and T1122C-but not F1116C, I1117C, I1119C, or L1120C-are accessible to externally applied MTS reagents [10]. Login to comment 123 ABCC7 p.Ser1118Cys ABCC7 p.Thr1121Cys ABCC7 p.Thr1115Cys ABCC7 p.Ile1112Cys Figure 3 confirms that application of external MTSES (200 bc;M) following channel activation with cAMP-stimulatory cocktail caused a decrease in whole cell current amplitude in T1115C, S1118C, and T1121C, but not in I1112C. Login to comment 124 ABCC7 p.Ser1118Cys Together, these results (Figs. 2b, 3b) suggest that S1118C represents the outermost extent of internal MTSES penetration into the pore, and T1115 the innermost extent of external MTSES penetration. Login to comment 127 ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys ABCC7 p.Thr1115Cys State-dependent accessibility of T1115C and S1118C in TM11 Modification of T1115C and S1118C by both internal and external MTSES is reminiscent of the accessibility pattern observed for TM6 cysteine mutant T338C [42]. Login to comment 128 ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys This similarity is perhaps not surprising since a disulfide bond can be formed between the two cysteine side chains of T338C and S1118C in open channels [43], indicating close physical proximity of these pore-exposed side chains. Login to comment 129 ABCC7 p.Thr338Cys Previously we suggested that the ability of T338C to be modified by MTSES from both sides of the membrane was due to this residue Fig. 5 Single channel currents carried by TM11 mutant channels. Login to comment 136 ABCC7 p.Glu1371Gln This suggestion was based in large part on the effects of the E1371Q mutation, which results in constitutively open CFTR channels, on side-dependent modification. Login to comment 137 ABCC7 p.Glu1371Gln ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys As shown in Fig. 4a, c, the E1371Q mutation significantly accelerated the rate of modification of both T1115C and S1118C by intracellular MTSES, suggesting that these cysteines are more readily modified from the inside in open channels. Login to comment 138 ABCC7 p.Glu1371Gln In contrast, these channels bearing the E1371Q mutation were almost completely insensitive to external MTSES (Fig. 4b), suggesting that these cysteines are much more difficult to modify from the outside in open channels (Fig. 4d). Login to comment 141 ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys This suggestion is consistent with data that S1118C can form a disulfide bond with T338C in open channels [43]. Login to comment 150 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala In addition, possible additive effects of reducing the side chain volumes of these two nearby residues was investigated using a T338A/S1118A double mutant. Login to comment 152 ABCC7 p.Ser1118Cys Previously it was shown that the S1118C mutation reduced single channel conductance at depolarized membrane potentials [10]. Login to comment 153 ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Val ABCC7 p.Ser1118Gln In contrast, S1118A had no effect on conductance, while S1118Q and S1118V were associated with Fig. 7 Thiocyanate permeability of mutants. Login to comment 155 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala The depolarizing (rightward) shift in the current reversal potential indicates an increased PSCN/PCl in the T338A/S1118A double mutant. Login to comment 156 ABCC7 p.Thr338Ala b Mean PSCN/PCl values calculated from reversal potential measurements under these conditions as described in the "Materials and methods" section. Mean of data from three to six patches. Asterisks indicate a significant difference from wild type (P<0.01), while hashtag indicates a significant difference from the T338A mutant (P<0.0002) Fig. 8 Acetate permeability of mutants. Login to comment 158 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala The hypolarizing (leftward) shift in the current reversal potential indicates an increased Pacetate/PCl in the T338A/S1118A double mutant. Login to comment 159 ABCC7 p.Thr338Ala b Mean Pacetate/PCl values calculated from reversal potential measurements under these conditions as described in the "Materials and methods" section. Mean of data from three to five patches. Asterisks indicate a significant difference from wild type, while hashtag indicates a significant difference from the T338A mutant (P<0.01) very small (<5 %) reductions in conductance (Fig. 5). Login to comment 160 ABCC7 p.Thr1115Ala Conductance was also unaltered in T1115A. Login to comment 161 ABCC7 p.Ser1118Cys In each case, the single channel I-V relationship remained linear (e.g., Fig. 5b), in contrast to the strongly inwardly rectified I-V relationship previously observed for S1118C [10]. Login to comment 162 ABCC7 p.Thr338Ala These very minor effects on conductance are in contrast with the large (>25 %) increase seen in T338A (Fig. 5), as reported previously [26]. Login to comment 163 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala The double mutant T338A/S1118A had a similarly elevated conductance that was not significantly different from that of T338A alone (P>0.75; Fig. 5). Login to comment 164 ABCC7 p.Thr338Ala Altered movement of permeant ions in the pore of T338A-CFTR is also reflected by changes in the voltage-dependent block of Cl-currents by low concentrations of permeant Au(CN)2 - ions [11, 14, 15]. Login to comment 165 ABCC7 p.Thr338Ala As shown in Fig. 6, channel block by intracellular Au(CN)2 - ions is altered in two distinct ways by the T338A mutation: (a) block is significantly weakened and (b) the I-V relationship in the presence of Au(CN)2 - shows an unusual "N"-shape (Fig. 6a), resulting in a "U"- shaped fractional current-voltage relationship (Fig. 6c) that indicates strongest block close to 0 mV membrane potential that is weakened at both more hyperpolarized and more depolarized voltages. Login to comment 166 ABCC7 p.Thr338Ala This unusual shape is thought to reflect increased unblock by blocker permeation at hyperpolarized voltages, which may reflect a reduced barrier to Au(CN)2 - movement inside the pore in T338A [11]. Login to comment 167 ABCC7 p.Ser1118Ala ABCC7 p.Thr1115Ala ABCC7 p.Ser1118Thr Block by intracellular Au(CN)2 - was also significantly weakened in S1118A, S1118T, and T1115A compared to wild type, especially at hyperpolarized membrane potentials (Fig. 6); however, no apparent "U"-shape to the fractional current-voltage relationship was observed (Fig. 6b). Login to comment 168 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Interestingly, block of the T338A/S1118A double mutant was slightly weaker than for T338A alone (Fig. 6c, d), suggesting that these two mutations might have additive effects on Au(CN)2 - binding in the pore. Login to comment 169 ABCC7 p.Ser1118Cys As observed at the single channel level (Fig. 5), the macroscopic I-V relationships for all mutants remained linear (e.g., Fig. 6a), in contrast to the strongly inwardly rectifying relationship previously observed for S1118C [10]. Login to comment 180 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala ABCC7 p.Thr1115Ala As proposed previously for TM6 residue T338 [42], the channel is shown as being in an "outward facing" configuration when closed (with T1115 and S1118 accessible from the outside), and switching to an "inward facing" configuration on opening (with T1115 and S1118 accessible from the inside) S1118A and T1115A, although this increase was much less than that observed in T338A (Fig. 7b). Login to comment 181 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Interestingly, SCN- permeability was further increased in the T338A/S1118A double mutant (Fig. 7). Login to comment 183 ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Val ABCC7 p.Ser1118Gln ABCC7 p.Thr1115Ala Acetate permeability was slightly increased in S1118A and T1115A, but not significantly changed in S1118Q or S1118V (Fig. 8b). Login to comment 184 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Again, the increase in acetate permeability seen in T338A was significantly augmented in the T338A/S1118A mutation (Fig. 8), suggesting an additive effect of these two mutations on organic anion permeability. Login to comment 187 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys ABCC7 p.Thr1122Cys 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 199 ABCC7 p.Thr338Cys ABCC7 p.Ser1118Cys ABCC7 p.Thr1115Cys Furthermore, using the same approach used previously to study side-dependent modification of T338C [42], we found that T1115C and S1118C were more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels (Fig. 4). Login to comment 200 ABCC7 p.Thr338Cys This pattern, which is common to that previously observed for T338C [42], is consistent with these residues showing alternating access to the extracellular and intracellular sides of the membrane during channel opening and closing (Fig. 9c). Login to comment 206 ABCC7 p.Thr338Ala However, mutagenesis of S1118 to residues with smaller (alanine) or larger (glutamine, valine) residues had surprisingly small effects on channel functional properties, in particular compared to those of mutagenesis of T338A. Login to comment 207 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala ABCC7 p.Thr1115Ala The effects of the S1118A mutation on permeant anion (Au(CN)2 - ) binding (Fig. 6), permeability of the lyotropic SCN- anion (Fig. 7), and permeability of the organic acetate anion (Fig. 8) were qualitatively similar to, but generally smaller than, those of T338A, and in fact similar effects were seen in T1115A. Login to comment 208 ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Val ABCC7 p.Ser1118Gln ABCC7 p.Thr1115Ala Neither S1118A nor T1115A significantly altered single channel conductance, although introduction of larger amino acid side chains in S1118Q and S1118V led to very small decreases in conductance (Fig. 5). Login to comment 209 ABCC7 p.Ser1118Phe Previously, it was shown that substitution of S1118 with a much larger phenylalanine residue (S1118F) caused a larger (~35 %) decrease in single channel conductance [46]. Login to comment 211 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Val ABCC7 p.Ser1118Gln ABCC7 p.Thr1115Ala Reduction of side chain volume in S1118A and T1115A, like T338A, led to an increase in the relative permeability of the small organic anion acetate, consistent with an increase in the apparent diameter of the narrowest region of the pore [25, 26]; however, introduction of side chains with larger volume (S1118Q, S1118V) did not lead to a decrease in acetate permeability (Fig. 8). Login to comment 213 ABCC7 p.Thr338Ala Simultaneous mutagenesis of T338 and S1118 to small alanine residues also had only small additional effects compared to the T338A mutation alone (Figs. 5, 6, 7, and 8). Login to comment 214 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Most striking here were a significantly increased permeability of the T338A/S1118A double mutant both to SCN- (Fig. 7) and to acetate (Fig. 8). Login to comment 215 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Permeability of small lyotropic anions like SCN- might be influenced by interactions throughout the pore [18, 38] or might be determined predominantly at a localized "selectivity filter" [18, 24] and so the apparently additive effects of the T338A and S1118A mutations is difficult to interpret in terms of the relative roles or locations of these two residues. Login to comment 216 ABCC7 p.Thr338Ala ABCC7 p.Ser1118Ala Permeability of large anions such as acetate is thought to be determined predominantly by steric factors at the narrowest part of the pore [25], and so the increase in acetate permeability in T338A/S1118A compared to either mutation alone might be considered evidence that these two mutations impact the dimensions of a common, narrow region of the pore. Login to comment 217 ABCC7 p.Ser1118Ala ABCC7 p.Thr1115Ala One possible reason contributing to the minor functional effects observed in these experiments is that S1118A (and T1115A) might be considered relatively conservative mutations leading to only small changes in amino acid side chain volume. Login to comment 218 ABCC7 p.Thr338Ala Nevertheless, T338A (which results in a similarly small reduction in side chain volume) is associated with much greater changes in pore properties. Login to comment 219 ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Cys ABCC7 p.Ser1118Gln Previously, the S1118C mutation was shown to decrease conductance at positive voltages, leading to inward rectification of both the single channel and macroscopic current-voltage relationships [10], and although this would be considered a very conservative mutation (one oxygen atom replaced by sulfur) this effect was not reproduced in S1118A, S1118Q or S1118 (Fig. 5). Login to comment 220 ABCC7 p.Ser1118Cys Given that the effects of the S1118C mutation on single channel conductance and rectification were exacerbated by modification by external MTSES, and mostly reversed by modification with positively charged MTSET [10], these effects might reflect partial negative charge of the introduced cysteine side chain rather than a change in side chain volume. Login to comment