ABCMdb

PMID: 23083715

El Hiani Y, Linsdell P
Tuning of CFTR chloride channel function by location of positive charges within the pore.
Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Lys95Gln The loss of conductance observed in the K95Q mutation was >50% rescued by substituting a lysine for each of five different pore-lining amino acids, suggesting that the exact location of the fixed positive charge is not crucial to support high conductance. Login to comment 3 ABCC7 p.Lys95Gln Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Login to comment 14 ABCC7 p.Ser1141Lys Thus, although neutralization of K95 causes a decrease in both Cl conductance and sensitivity to cytoplasmic open-channel blockers, these changes in channel function can be reversed by concurrent mutagenesis of a serine residue in TM12 to lysine (the S1141K mutant), suggesting that these two nearby residues lining the inner vestibule are functionally interchangeable (8). Login to comment 15 ABCC7 p.Ser1141Lys Interestingly, using the S1141K mutation to introduce a second positive charge to the inner vestibule did not increase Cl conductance (suggesting that a single positive charge is sufficient to maximize conductance), but instead conferred strong block by cytoplasmically applied multivalent anions (leading to an overall decrease in channel function) (8). Login to comment 27 ABCC7 p.Lys95Gln Lysine mutations were made in both wild-type (WT) and K95Q backgrounds, resulting in channel constructs that contained two or one positively charged lysine residues in the putative inner vestibule of the pore, respectively. Login to comment 42 ABCC7 p.Lys95Gln This requirement is clear in K95Q, which reduces conductance to ~15% of WT (Fig. 2, A-C). Login to comment 43 ABCC7 p.Met348Lys ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Ala349Lys After neutralization of this endogenous positive charge by the K95Q mutation, introduction of a positive charge at other sites (by mutagenesis to lysine) caused a significant increase in unitary conductance to between 51 5 1% (in K95Q/A349K; n &#bc; 10) and 77 5 1% (in K95Q/M348K; n &#bc; 12) of WT conductance (Fig. 2, A-C), suggesting that a positive charge located at other positions in the pore can effectively rescue the WT conductance phenotype. Login to comment 49 ABCC7 p.Gln98Lys ABCC7 p.Val345Lys conductance (Fig. 2, B and C), especially in Q98K (conductance 75 5 1% of WT, n &#bc; 6) and V345K (64 5 3% of WT, n &#bc; 9), and in no case was conductance increased by the addition of a second positive charge. Login to comment 50 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln As a result, the effects of removing the native positive charge by introducing the K95Q mutation appear highly background specific (Fig. 2 D), i.e., the effects of the K95Q mutation on conductance are far more dramatic in a WT background than when an additional positive charge is present. Login to comment 54 ABCC7 p.Lys95Gln As shown in Fig. 3, addition of 50 mM NPPB to the cytoplasmic side of inside-out patches caused strong inhibition of WT CFTR but had no noticeable effect on K95Q channels. Login to comment 55 ABCC7 p.Met348Lys ABCC7 p.Lys95Gln ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Additional mutations in a K95Q background to transplant the positive charge to pore-lining positions in TM1 (Q98K) or TM6 (I344K, V345K, M348K, and A349K) partially restored NPPB block (Fig. 3), although in no case was the block as strong as for the WT. Login to comment 56 ABCC7 p.Met348Lys ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys The rank order of the apparent strength of NPPB block was WT > K95Q/V345K > K95Q/I344K > K95Q/Q98K ~ K95Q/ M348K ~ K95Q/A349K (Fig. 3 B). Login to comment 57 ABCC7 p.Lys95Ser ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys Similar results were previously reported for NPPB block of K95S/S341K and K95S/S1141K (8). Login to comment 58 ABCC7 p.Ser1141Lys A striking and interesting effect of adding a second positive charge to the inner vestibule, previously demonstrated in S1141K (8), was a dramatic increase in susceptibility to block by polyvalent anions present in the cytoplasmic solution. Login to comment 60 ABCC7 p.Met348Lys ABCC7 p.Ser341Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys As shown in Fig. 4, block by Pt(NO2)4 2 was significantly strengthened in each of the mutants Q98K, I344K, V345K, M348K, and A349K, as well as in the previously unstudied S341K. Login to comment 61 ABCC7 p.Met348Lys ABCC7 p.Ser341Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys At 0 mV membrane potential, the apparent Kd for Pt(NO2)4 2 block was in the rank order V345K (3.3 5 0.9 mM, n &#bc; 7) % I344K (4.5 5 0.7 mM, n &#bc; 6) < S341K (26.6 5 1.8 mM, n &#bc; 7) < M348K (80.9 5 7.2 mM, n &#bc; 5) % Q98K (95.4 5 11.0 mM, n &#bc; 6) % A349K (117.4 5 7.7 mM, FIGURE 2 Single-channel conductance is restored by moving the positive charge from K95. Login to comment 64 ABCC7 p.Lys95Gln Note that the reduced single-channel current amplitude of K95Q is rescued by concurrent mutagenesis of other amino acids (Q98, I344, V345, M348, and A349) to lysine. Login to comment 66 ABCC7 p.Lys95Gln The leftmost panel shows WT (green) and K95Q (red). Login to comment 67 ABCC7 p.Lys95Gln The other five panels show the effects of the indicated lysine-introducing mutations in either a WT (open green symbols) or K95Q (open red symbols) background. Login to comment 68 ABCC7 p.Lys95Gln In each case, the lines fitted to WT and K95Q data are indicated in green and red, respectively, as a reference. Login to comment 70 ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln *Significant difference from WT (p < 0.05); # significant difference from K95Q (p < 1010 ); y significant difference from the same lysine mutation in a K95Q background (p < 0.05). Login to comment 72 ABCC7 p.Lys95Gln In each case, the effect of the K95Q mutation was significantly reduced by the presence of an introduced lysine at other positions (*p < 1010 compared with WT). Login to comment 74 ABCC7 p.Met348Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys Blocker voltage dependence was also significantly changed in most mutants, with the effective blocker valence (zd) being significantly increased in M348K and significantly decreased in Q98K, V345K, and A349K (Fig. 4 F). Login to comment 78 ABCC7 p.Val345His ABCC7 p.Val345His ABCC7 p.Ile344His ABCC7 p.Ile344His As shown in Fig. 5, block of both I344H and V345H by Pt(NO2)4 2 was drastically stronger at pH 5.5 than at pH 9.0, with the mean Kd(0) for I344H and V345H being ~13-fold and ~38-fold lower, respectively, at acid pH (Fig. 5 D). Login to comment 80 ABCC7 p.Val345His ABCC7 p.Ile344His In contrast to these large effects of pH on block by Pt(NO2)4 2 , the single-channel conductance of I344H and V345H, as well as that of the WT, were not significantly different at pH 5.5 and pH 9.0 (Fig. 6). Login to comment 81 ABCC7 p.Val345His ABCC7 p.Ile344His Whereas the conductance of V345H was reduced to ~80% of WT conductance (independently of pH), the conductance of I344H was not significantly different from that of the WT (Fig. 6 C). Login to comment 82 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys ABCC7 p.Val345His ABCC7 p.Ile344His Interestingly, whereas the conductance of I344H (at pH 5.5) was not significantly different from that of I344K (p > 0.37; Fig. 2 C), the conductance of V345H (pH 5.5) was ~20% greater than that of V345K (p < 0.002). Login to comment 85 ABCC7 p.Lys95Gln Transplanting the charge to other sites in TM1 (Q98) and TM6 (I344, V345, M348, and A349; Fig. 2), as well as S1141 in TM12 (8), results in restoration of at least 50% of the loss of conductance caused by the K95Q mutation. Login to comment 86 ABCC7 p.Ser341Lys Only S341K, located more deeply in the pore from its cytoplasmic end, was unable to support high conductance (8). Login to comment 88 ABCC7 p.Lys95Gln Although this residue does exert some influence over channel conductance (7), it presumably cannot compensate for the loss of positive charge in K95Q. Login to comment 90 ABCC7 p.Met348Lys ABCC7 p.Ser1141Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys However, although a single positive charge is necessary, the addition of a second positive charge to this region of the pore (as in the point mutants Q98K, I344K, V345K, M348K, and A349K) failed to increase conductance above WT levels (Fig. 2), as previously observed for S1141K (8). Login to comment 96 ABCC7 p.Lys95Gln *Significant difference from WT (p < 0.005); # significant difference from K95Q (p < 0.002). Login to comment 99 ABCC7 p.Met348Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys In fact, the addition of a second positive charge in Q98K, I344K, V345K, M348K, and A349K led to a small, but significant, decrease in conductance (Fig. 2 C). Login to comment 102 ABCC7 p.Val345His ABCC7 p.Ile344His In fact, the results of Fig. 6 support the latter explanation, because specifically toggling the positive charge in I344H or V345H by changing the pH did not alter single-channel conductance. Login to comment 103 ABCC7 p.Val345Lys ABCC7 p.Val345His Furthermore, the notion that charge is not the only factor that influences conductance is supported by the fact that the conductance of V345K was significantly less than that of V345H when measured at pH 5.5, where the histidine side chain is expected to bear a positive charge. Login to comment 105 ABCC7 p.Met348Lys ABCC7 p.Lys95Gln ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys The weakening of blocker binding seen in K95Q is partially reversed by the second site mutations I344K and V345K, and to a lesser extent Q98K, M348K, and A349K. Login to comment 106 ABCC7 p.Lys95Ser ABCC7 p.Ser1141Lys It was previously reported that the double mutant K95S/S1141K showed slightly increased potency of NPPB block compared with WT (8). Login to comment 126 ABCC7 p.Lys95Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys ABCC7 p.Gln98Cys This relative location of amino acids is also supported by experimental evidence that disulfide bonds can be formed between cysteine side chains substituted for K95 and S1141 (8), as well as between K95C and I344C, and between Q98C and I344C (13). Login to comment 128 ABCC7 p.Val345His (A) Example leak-subtracted macroscopic I/V relationships for V345H at intracellular pH of 5.5 (left) or 9.0 (right, different patch). Login to comment 130 ABCC7 p.Val345His ABCC7 p.Ile344His (B) Mean concentration-inhibition relationships for WT (circles), I344H (squares), and V345H (triangles) at intracellular pH 5.5 (left, solid symbols) or 9.0 (right, open symbols) at a membrane potential of 100 mV. Each relationship was fitted according to Eq. 1. Login to comment 138 ABCC7 p.Val345His (A) Example single-channel currents carried by V345H at a membrane potential of 50 mV, at a bath pH of 5.5 (left) or 9.0 (right, different patch). Login to comment 140 ABCC7 p.Val345His (B) Mean single-channel i/V relationships for WT and V345H under these two pH conditions. Login to comment 146 ABCC7 p.Met348Lys ABCC7 p.Ser1141Lys ABCC7 p.Ser341Lys ABCC7 p.Ala349Lys ABCC7 p.Gln98Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Again this appears to be a relatively nonsite-specific effect of positive charge, since all mutants studied (Q98K, S341K, I344K, V345K, M348K, and A349K) led to significant increase in apparent affinity of Pt(NO2)4 2 block (Fig. 4), as did S1141K (8). Login to comment 147 ABCC7 p.Val345Lys ABCC7 p.Ile344Lys Most striking were I344K and V345K, which led to an ~70-fold and ~95-fold increase in apparent affinity, respectively (Fig. 4 E). Login to comment 148 ABCC7 p.Val345His ABCC7 p.Ile344His At these two sites, the effect on Pt(NO2)4 2 block could be ascribed unambiguously to an effect of the introduced positive charge, because the mutants I344H and V345H showed Pt(NO2)4 2 block similar to that of WT at pH 9.0, where the histidine side chains are expected to be uncharged, whereas at pH 5.5, which should promote protonation of these side chains, Pt(NO2)4 2 block was drastically strengthened (Fig. 5). Login to comment 151 ABCC7 p.Ser1141Lys ABCC7 p.Val345Lys ABCC7 p.Ile344Lys The cartoon model of Fig. 1 suggests that it is proximity to the endogenous positive charge at K95, at least in terms of location along the axis of the pore, that determines the ability of introduced positive charges to strengthen Pt(NO2)4 2 block, since I344K and V345K (Fig. 4), together with S1141K (8), give the most potent block. Login to comment 157 ABCC7 p.Ser341Lys At the outermost extent of this inner pore region, the open-channel pore may become too narrow to accommodate a positive charge (as previously proposed to explain the low single-channel conductance of S341K and other mutations at the same site (8)). Login to comment 158 ABCC7 p.Lys95Gln ABCC7 p.Ala349Lys At its cytoplasmic entrance, the pore is wider (15,22), which may explain the weaker ability of the A349K mutation nearer the cytoplasmic end of the inner vestibule to restore single-channel conductance in a K95Q background (Fig. 2). Login to comment