ABCMdb

ABCC7 p.Val345Lys

Predicted by SNAP2:	A: N (61%), C: D (59%), D: D (85%), E: D (80%), F: D (85%), G: D (80%), H: D (85%), I: D (53%), K: D (85%), L: D (63%), M: D (80%), N: D (75%), P: D (66%), Q: D (80%), R: D (80%), S: D (59%), T: D (53%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: N, T: N, W: D, Y: D,

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[hide] Tuning of CFTR chloride channel function by locati... Biophys J. 2012 Oct 17;103(8):1719-26. doi: 10.1016/j.bpj.2012.09.020. Epub 2012 Oct 16. 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., [PMID:23083715]

Abstract [show]
High unitary Cl(-) conductance in the cystic fibrosis transmembrane conductance regulator Cl(-) channel requires a functionally unique, positively charged lysine residue (K95) in the inner vestibule of the channel pore. Here we used a mutagenic approach to investigate the ability of other sites in the pore to host this important positive charge. 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. Moving the positive charge also restored open-channel blocker interactions that are lost in K95Q. Introducing a second positive charge in addition to that at K95 did not increase conductance at any site, but did result in a striking increase in the strength of block by divalent Pt(NO(2))(4)(2-) ions. Based on the site dependence of these effects, we propose that although the exact location of the positive charge is not crucial for normal pore properties, transplanting this charge to other sites results in a diminution of its effectiveness that appears to depend on its location along the axis of the pore.

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49 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
55 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 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
60 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 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
74 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
82 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
90 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
99 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
103 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 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
146 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 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
151 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

[hide] State-dependent blocker interactions with the CFTR... Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28. Linsdell P
State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.
Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28., [PMID:24671572]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

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6 Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4 2- altered blocker effects both on Cl-permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4 2- interaction with a single site within the pore. Login to comment
31 Furthermore, when the number of positive charges lining this part of the inner vestibule is increased from one (as in wild type) to two (for example in the channel mutants I344K, V345K, and S1141K), blocker potency is increased [8, 36]. Login to comment
43 Where the properties of different channel pore variants (wild type, K95Q, I344K, V345K) have been directly compared in wild type and E1371Q backgrounds, the wild-type background is referred to as "1371E" to indicate that the endogenous glutamate residue is present at this position. Login to comment
109 Removal of the key endogenous positive charge (using the K95Q mutation) greatly weakens Pt(NO2)4 2- block (Fig. 3(B-D)), whereas addition of a second pore-lining positive charge (in I344K or V345K) dramatically strengthens open-channel block (Fig. 3(B-D)). Login to comment
110 Interestingly, not only blocker binding affinity (Fig. 3(D)) but also blocker voltage dependence (Fig. 3(E)) appeared correlated with the number of positively charged lysine side chains lining the inner vestibule of the pore; both I344K/E1371Q and V345K/E1371Q gave very strong, very strongly voltage-dependent block (Fig. 3(A, C-E)). Login to comment
113 In contrast, the strong inhibitory effects of Pt(NO2)4 2- on both I344K- and V345K-containing channels were observed in both E1371Q and 1371E backgrounds (Fig. 4(B)). Login to comment
114 Most strikingly, however, the very strong voltage dependence of block seen in both I344K/E1371Q and V345K/E1371Q (Fig. 3) was not observed in I344K/1371E and V345K/1371E channels that were allowed to open and close normally; instead, block was very strong but almost completely voltage-independent (Fig. 4). Login to comment
115 In many ways, these results with I344K and V345K recapitulate the results observed when comparing wild type and E1371Q channels (Fig. 1); block is weaker in E1371Q-containing channels (Fig. 4(C)), and blocker voltage dependence is greatly decreased (Fig. 4(D)). Login to comment
119 Gating of I344K and V345K channels was affected by Pt(NO2)4 2- in a similar manner to those of wild-type channels, but at much lower concentrations. Login to comment
122 Investigation of the effect of different concentrations of Pt(NO2)4 2- on the PO of wild type, I344K and V345K channels (Fig. 5(C)) confirms a dramatic increase in the apparent potency with which Pt(NO2)4 2- ions interact with these channel pore mutants. Login to comment
123 Effect of extracellular Clon Pt(NO2)4 2- block As a consequence of the striking difference in voltage-dependence of block of I344K and V345K-containing channels studied in 1371E (Fig. 4) or E1371Q backgrounds (Fig. 3), there was an unusual "cross-over" effect in the relationship between the KD for Pt(NO2)4 2- block and voltage (Fig. 4(B)), suggesting that block is stronger in 1371E than in E1371Q channels at positive voltages, but weaker in 1371E at the most negative voltages studied. Login to comment
126 Under these conditions, cross-over of the KD-voltage relationship was observed for wild type and I344K channels, whereas for V345K channels block was now stronger in E1371Q channels at all voltages studied. Login to comment
135 Under these ionic conditions, the E1371Q mutation increased the apparent affinity of block (Fig. 7(A, B)), an effect that was especially strong in I344K and V345K channels and at hyperpolarized voltages. Login to comment
136 This contrasts with what was found under high [Cl- ] conditions, where the E1371Q mutation decreased the affinity of block, especially in I344K and V345K (Fig. 4(C)). Login to comment
146 However, [Cl- ] has little effect on the voltage dependence of block (Fig. 7(E)), which is already very strong in I344K/E1371Q and V345K/E1371Q channels (Figs. 4(D) and 7(C)). Login to comment
148 The strong effect of [Cl- ] on the strength of block of I344K and V345K is abolished (Fig. 7(D)), and high [Cl- ] actually reduces the strong apparent voltage dependence of block in these same mutants (Fig. 7(E)). Login to comment
150 (A) Example currents recorded from inside out patches each containing two active CFTR channels, either I344K (top) or V345K (bottom). Login to comment
152 All closed state is indicated by the line to the left. Note that V345K exhibits a significantly reduced single-channel current amplitude, as described and quantified previously [8]. Login to comment
155 (C) Effect of different concentrations of Pt(NO2)4 2- on the PO of wild type, I344K and V345K channels as indicated. Login to comment
165 Mean of data from 5 to 11 patches Pt(NO2)4 2-binding within the inner vestibule of the pore, since they are weakened by the K95Q mutation and strengthened in I344K and V345K (Fig. 3(C)), as described previously [8, 36]. Login to comment
172 Thus, effects on gating are apparently lost in K95Q (Fig. 4(B)), and are substantially strengthened by mutations (I344K, V345K) that increase the strength of binding inside the pore (Figs. 4 and 5). Login to comment
175 However, these two effects appear unable to explain the overall effects of Pt(NO2)4 2- in I344K and V345K (Figs. 4(B) and 6(C)), or effects on wild type under low extracellular [Cl- ] conditions (Fig. 6(C)). Login to comment
187 Perhaps supporting the former hypothesis, this putative stimulatory effect of Pt(NO2)4 2- does appear to be positively correlated with strong open-channel block, i.e., it is not observed in K95Q but is prominent in I344K and V345K channels that show strong binding, and at negative voltages that promote open-channel block (Fig. 6). Login to comment
200 Interestingly, this apparent knock-off effect is greatly strengthened in I344K/E1371Q and V345K/E1371Q (Fig. 7(D)), suggesting Fig. 8 Schematic summary of the effects of Pt(NO2)4 2- . Login to comment

[hide] Location of a permeant anion binding site in the c... J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12. Rubaiy HN, Linsdell P
Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12., [PMID:25673337]

Abstract [show]
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

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79 e Mean KD values for Au(CN)2 - block for these channel constructs, as well as the additional positive charge mutants V345K/ E1371Q and S1141K/E1371Q, obtained as described in Fig. 1. f Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and the expected number of fixed positive charges in the pore inner vestibule in different channel constructs. Login to comment
90 Consistent with this, mutation of any of these three residues to lysine (I344K, V345K, S1141K) led to a significant strengthening of Au(CN)2 - block (Fig. 3d-f), with mean KD values at -100 mV being reduced by 18-fold (I344K/E1371Q), 17-fold (V345K/E1371Q) and 7-fold (S1141K/E1371Q), respectively. Login to comment