ABCC7 p.Ile344Lys
| Predicted by SNAP2: | A: N (66%), C: N (66%), D: D (59%), E: N (57%), F: N (53%), G: D (53%), H: D (53%), K: N (57%), L: N (78%), M: N (82%), N: N (57%), P: N (53%), Q: N (61%), R: N (66%), S: N (72%), T: N (66%), V: N (82%), W: D (66%), Y: D (53%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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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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No. Sentence 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.
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ABCC7 p.Ile344Lys 23083715:55:123
status: NEW56 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).
X
ABCC7 p.Ile344Lys 23083715:56:81
status: NEW60 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.
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ABCC7 p.Ile344Lys 23083715:60:101
status: NEW61 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.
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ABCC7 p.Ile344Lys 23083715:61:122
status: NEW82 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).
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ABCC7 p.Ile344Lys 23083715:82:105
status: NEW90 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).
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ABCC7 p.Ile344Lys 23083715:90:156
status: NEW99 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).
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ABCC7 p.Ile344Lys 23083715:99:59
status: NEW105 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.
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ABCC7 p.Ile344Lys 23083715:105:97
status: NEW146 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).
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ABCC7 p.Ile344Lys 23083715:146:122
status: NEW147 Most striking were I344K and V345K, which led to an ~70-fold and ~95-fold increase in apparent affinity, respectively (Fig. 4 E).
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ABCC7 p.Ile344Lys 23083715:147:19
status: NEW151 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.
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ABCC7 p.Ile344Lys 23083715:151:256
status: NEW[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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None has been submitted yet.
No. Sentence Comment
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.
X
ABCC7 p.Ile344Lys 24671572:6:75
status: NEW31 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].
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ABCC7 p.Ile344Lys 24671572:31:172
status: NEW43 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.
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ABCC7 p.Ile344Lys 24671572:43:74
status: NEW109 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)).
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ABCC7 p.Ile344Lys 24671572:109:182
status: NEW110 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)).
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ABCC7 p.Ile344Lys 24671572:110:231
status: NEW113 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)).
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ABCC7 p.Ile344Lys 24671572:113:66
status: NEW114 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).
X
ABCC7 p.Ile344Lys 24671572:114:83
status: NEWX
ABCC7 p.Ile344Lys 24671572:114:142
status: NEW115 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)).
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ABCC7 p.Ile344Lys 24671572:115:33
status: NEW119 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.
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ABCC7 p.Ile344Lys 24671572:119:10
status: NEW122 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.
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ABCC7 p.Ile344Lys 24671572:122:95
status: NEW123 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.
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ABCC7 p.Ile344Lys 24671572:123:125
status: NEW126 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.
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ABCC7 p.Ile344Lys 24671572:126:97
status: NEW135 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.
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ABCC7 p.Ile344Lys 24671572:135:147
status: NEW136 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)).
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ABCC7 p.Ile344Lys 24671572:136:138
status: NEW146 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)).
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ABCC7 p.Ile344Lys 24671572:146:114
status: NEW148 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)).
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ABCC7 p.Ile344Lys 24671572:148:56
status: NEW150 (A) Example currents recorded from inside out patches each containing two active CFTR channels, either I344K (top) or V345K (bottom).
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ABCC7 p.Ile344Lys 24671572:150:103
status: NEW155 (C) Effect of different concentrations of Pt(NO2)4 2- on the PO of wild type, I344K and V345K channels as indicated.
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ABCC7 p.Ile344Lys 24671572:155:78
status: NEW165 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].
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ABCC7 p.Ile344Lys 24671572:165:159
status: NEW172 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).
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ABCC7 p.Ile344Lys 24671572:172:114
status: NEW175 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)).
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ABCC7 p.Ile344Lys 24671572:175:90
status: NEW187 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).
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ABCC7 p.Ile344Lys 24671572:187:215
status: NEW200 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- .
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ABCC7 p.Ile344Lys 24671572:200:73
status: NEW[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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None has been submitted yet.
No. Sentence Comment
78 d Example macroscopic I-V relationships for I344K/E1371Q (left) and K95Q/I344K/E1371Q (right) CFTR channels recorded before (control) and after the addition of a low concentration (10 lM) of Au(CN)2 - to the intracellular (bath) solution.
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ABCC7 p.Ile344Lys 25673337:78:44
status: NEWX
ABCC7 p.Ile344Lys 25673337:78:73
status: NEW90 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.
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ABCC7 p.Ile344Lys 25673337:90:73
status: NEWX
ABCC7 p.Ile344Lys 25673337:90:219
status: NEW91 ''Moving`` the key positive charge from TM1 to TM6 by mutagenesis (in the K95Q/I344K/E1371Q mutant) resulted in Au(CN)2 - block that was intermediate in strength between E1371Q and I344K/E1371Q (Fig. 3e-f).
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ABCC7 p.Ile344Lys 25673337:91:79
status: NEWX
ABCC7 p.Ile344Lys 25673337:91:181
status: NEW103 In contrast, neither K95Q nor I344K altered the permeability selectivity sequence; in fact, the only significant difference between either K95Q/ E1371Q or I344K/E1371Q compared to E1371Q was a small increase in PF/PCl in K95Q/E1371Q.
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ABCC7 p.Ile344Lys 25673337:103:30
status: NEWX
ABCC7 p.Ile344Lys 25673337:103:155
status: NEW109 Note that the normal lyotropic relationship between relative permeability and Gh is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q.
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ABCC7 p.Ile344Lys 25673337:109:148
status: NEW[hide] Interactions between permeant and blocking anions ... Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17. Linsdell P
Interactions between permeant and blocking anions inside the CFTR chloride channel pore.
Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17., [PMID:25892339]
Abstract [show]
Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.
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None has been submitted yet.
No. Sentence Comment
3 A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4 2-binding affinity also greatly strengthened antagonistic Cl- :blocker interactions as well as the voltage-dependence of block.
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ABCC7 p.Ile344Lys 25892339:3:35
status: NEW4 Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4 2- ions but also with extracellular Cl- , and that altered blocker Cl- - and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl-binding in the outer vestibule.
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ABCC7 p.Ile344Lys 25892339:4:65
status: NEW6 However, the I344K mutation does not increase single channel conductance following disruption of Cl-binding in the outer vestibule in R334Q channels.
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ABCC7 p.Ile344Lys 25892339:6:13
status: NEW99 Thus, K95Q/E1371Q is associated with weakened Pt(NO2)4 2- block (Fig. 4A-E), and I344K/E1371Q with greatly strengthened block (Fig. 4F-J).
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ABCC7 p.Ile344Lys 25892339:99:81
status: NEW101 Thus Pt KD was only very weakly [Cl- ]o-sensitive in K95Q/E1371Q (Fig. 4E), but very strongly [Cl- ]o-dependent in I344K/E1371Q (Fig. 4J).
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ABCC7 p.Ile344Lys 25892339:101:115
status: NEW102 The affinity, voltage dependence, and [Cl- ]o-dependence of block in E1371Q, K95Q/E1371Q and I344K/E1371Q are compared directly in Fig. 5 and Table 1.
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ABCC7 p.Ile344Lys 25892339:102:93
status: NEW109 Since the R334Q and I344K mutations in different parts of the pore (Fig. 1) had opposite effects on the [Cl- ]o-sensitivity of Pt(NO2)4 2- block - which was practically abolished in R334Q/E1371Q (Fig. 6) but greatly increased in I344K/E1371Q (Figs. 4, 5) - these mutations were combined to generate a R334Q/I344K/E1371Q mutant.
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ABCC7 p.Ile344Lys 25892339:109:20
status: NEWX
ABCC7 p.Ile344Lys 25892339:109:229
status: NEWX
ABCC7 p.Ile344Lys 25892339:109:307
status: NEW110 Not only did R334Q/I344K/E1371Q channels exhibit strong Pt(NO2)4 2- block (Table 1), but block was also strongly dependent on extracellular [Cl- ] (Fig. 6E-H).
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ABCC7 p.Ile344Lys 25892339:110:19
status: NEW111 This confirms that the I344K mutation not only strengthens interactions with intracellular Pt(NO2)4 2- ions but also with extracellular Cl-ions. Furthermore, this effect on interactions with extracellular Cl- appears independent of the presence of a positively charged side chain at position 334: whereas the R334Q mutation abolishes Cl- -dependence of block in E1371Q channels (Fig. 6D), it does not in I344K- bearing E1371Q channels (Fig. 6H).
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ABCC7 p.Ile344Lys 25892339:111:23
status: NEWX
ABCC7 p.Ile344Lys 25892339:111:404
status: NEW112 Thus, interactions with external Cl- that are lost in R334Q/E1371Q channels are at least partially restored by the I344K mutation.
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ABCC7 p.Ile344Lys 25892339:112:115
status: NEW113 To examine if the I344K mutation also resulted in altered interactions with other extracellular anions - and if this effect was also independent of R334 -Pt(NO2)4 2- block was also investigated with other extracellular anions, both in I344K/E1371Q and in R334Q/I344K/ E1371Q channels (Fig. 7).
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ABCC7 p.Ile344Lys 25892339:113:18
status: NEWX
ABCC7 p.Ile344Lys 25892339:113:235
status: NEWX
ABCC7 p.Ile344Lys 25892339:113:261
status: NEW116 First, SCN- had a very strong negative effect on Pt(NO2)4 2- block in I344K/E1371Q, increasing Pt KD(0) N600-fold relative to that with gluconate and N25-fold relative to that with Cl- (Fig. 7C, E, G).
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ABCC7 p.Ile344Lys 25892339:116:70
status: NEW117 Secondly, formate was able to significantly increase Pt KD(0) relative to gluconate in both I344K/E1371Q (Fig. 7G) and in R334Q/I344K/E1371Q (Fig. 7H), unlike in E1371Q where formate had no significant effect (Fig. 3D, E).
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ABCC7 p.Ile344Lys 25892339:117:92
status: NEWX
ABCC7 p.Ile344Lys 25892339:117:128
status: NEW128 Pt KD(0) (4 mM Cl- ) (bc;M) zb4; (4 mM Cl- ) Pt KD(0) (154 mM Cl- ) (bc;M) zb4; (154 mM Cl- ) E1371Q 183.7 &#b1; 33.2 (9) -0.397 &#b1; 0.030 (9) 441.0 &#b1; 28.6 (7) -0.503 &#b1; 0.026 (7) R899Q/E1371Q 189.9 &#b1; 55.0 (6) -0.362 &#b1; 0.063 (6) 434.0 &#b1; 32.2 (7) -0.458 &#b1; 0.048 (7) K95Q/E1371Q 1110 &#b1; 172 (6)** -0.244 &#b1; 0.022 (6)* 1422 &#b1; 218 (6)** -0.193 &#b1; 0.041 (6)** I344K/E1371Q 6.92 &#b1; 1.48 (6)** -1.589 &#b1; 0.125 (6)** 164.7 &#b1; 27.5 (7)** -1.604 &#b1; 0.080 (7)** R334Q/E1371Q 1081 &#b1; 220 (4)** -0.637 &#b1; 0.106 (4)* 1112 &#b1; 144 (4)** -0.621 &#b1; 0.051 (4)* R334Q/I344K/E1371Q 39.24 &#b1; 7.94 (4)* -1.093 &#b1; 0.037 (4)** 258.3 &#b1; 30.7 (5)* -1.075 &#b1; 0.033 (5)** Fig. 4. Effect of mutations that weaken or strengthen intracellular Pt(NO2)4 2- block.
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ABCC7 p.Ile344Lys 25892339:128:405
status: NEWX
ABCC7 p.Ile344Lys 25892339:128:622
status: NEW129 (A, B, F, G) Example macroscopic I-V relationships for K95Q/E1371Q (A, B) or I344K/E1371Q (F, G) under high (154 mM; A, F) or low (4 mM; B, G) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution.
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ABCC7 p.Ile344Lys 25892339:129:77
status: NEW140 Quantitative analysis of anion binding in mutant channels Similar analysis of Cl- -dependent Pt(NO2)4 2- block in K95Q/E1371Q and I344K/E1371Q (Fig. 8B, C) provides additional insight into the effect of mutations close to the putative Pt(NO2)4 2-binding site.
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ABCC7 p.Ile344Lys 25892339:140:130
status: NEW145 The I344K mutant has a profound impact on channel binding affinity of both intracellular Pt(NO2)4 2- (Fig. 8D, E) and extracellular Cl- (Fig. 8F) (summarized in Table 2).
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ABCC7 p.Ile344Lys 25892339:145:4
status: NEW146 Compared to E1371Q, the intrinsic Pt(NO2)4 2-binding affinity of I344K/E1371Q is increased ~1400-fold by the addition of a second fixed positive charge in the inner vestibule, with no apparent change in the voltage dependence of binding (Fig. 8D).
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ABCC7 p.Ile344Lys 25892339:146:65
status: NEW148 In fact, the impact of Cl-binding on Pt(NO2)4 2-binding affinity (the difference between Pt Kvac and Pt Kocc) appears much greater in I344K/E1371Q than in E1371Q (Fig. 9).
X
ABCC7 p.Ile344Lys 25892339:148:134
status: NEW149 Finally, Cl-binding affinity is higher in I334K/E1371Q (Fig. 8F; Table 2), suggesting that the I344K mutation strengthens interactions with external Cl-ions as well as with internal Pt(NO2)4 2- ions. Furthermore, the voltage-dependence of Cl-binding is greatly increased, with Cl zb4; being increased N5-fold (Fig. 8F), suggesting that external Cl-ions cross a much larger part of the transmembrane electric field to reach their binding site in this mutant.
X
ABCC7 p.Ile344Lys 25892339:149:95
status: NEW154 Although the R334Q mutant abolished Cl- -dependence of Pt(NO2)4 2- block, the double pore mutant R334Q/I344K/E1371Q showed strongly Cl- -dependent block (Fig. 6E-H; Fig. 10B), suggesting that the presence of the I344K mutant restores Cl-binding that is lost in R334Q/E1371Q.
X
ABCC7 p.Ile344Lys 25892339:154:103
status: NEWX
ABCC7 p.Ile344Lys 25892339:154:212
status: NEW155 Quantitative analysis of data from R334Q/I344K/ E1371Q (Fig. 10; Table 2) suggests that the presence of the second fixed positive charge in the inner vestibule at position 344 still supports strong Pt(NO2)4 2-binding (Fig. 10C), although (as for E1371Q) Pt(NO2)4 2-binding is somewhat weakened by the R334Q mutation (Fig. 10C).
X
ABCC7 p.Ile344Lys 25892339:155:41
status: NEW156 Binding of Pt(NO2)4 2-to Cl- -occupied channels is similar in R334Q/E1371Q and R334Q/I344K/E1371Q (Fig. 10D), suggesting that R334 plays little or no role in ion:ion interactions in mutant channels bearing a positive charge at position 344.
X
ABCC7 p.Ile344Lys 25892339:156:85
status: NEW157 Chloride binding also remains strong in R334Q/I344K/E1371Q, although it too is somewhat weakened by the R334Q mutation (Fig. 10E).
X
ABCC7 p.Ile344Lys 25892339:157:46
status: NEW158 The strong effect of external Clon Pt(NO2)4 2-binding to R334Q/I344K/E1371Q channels is illustrated in Fig. 9D.
X
ABCC7 p.Ile344Lys 25892339:158:63
status: NEW159 Because the effect of external SCN- on Pt(NO2)4 2- block was so dramatically altered in I344K/E1371Q (Fig. 7C, E, G), quantitative analysis of the effects of SCN- was also carried out (Fig. 11; Table 3).
X
ABCC7 p.Ile344Lys 25892339:159:88
status: NEW160 As for external Cl- (Fig. 8), there was a linear relationship between [SCN- ]o and Pt KD, both in E1371Q (Fig. 11A) and I344K/E1371Q (Fig. 11E), consistent with competition between SCN- and Pt(NO2)4 2- ions.
X
ABCC7 p.Ile344Lys 25892339:160:120
status: NEW161 In I344K/ Fig. 5. Effect of mutations at the putative blocker binding site on [Cl- ]-dependence of block.
X
ABCC7 p.Ile344Lys 25892339:161:3
status: NEW162 (A) Relationship between Pt(NO2)4 2-binding affinity (Pt KD at 0 mV) and [Cl- ]o for E1371Q (black), K95Q/E1371Q (blue; see Fig. 1) and I344K/E1371Q (red; see Fig. 1).
X
ABCC7 p.Ile344Lys 25892339:162:136
status: NEW168 (A, B, E, F) Example macroscopic I-V relationships for R334Q/E1371Q (A, B) or R334Q/I344K/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution.
X
ABCC7 p.Ile344Lys 25892339:168:84
status: NEW173 Dotted lines indicate the fit to data for E1371Q (see Fig. 2H) or I344K/E1371Q (see Fig. 4J) as indicated.
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ABCC7 p.Ile344Lys 25892339:173:66
status: NEW176 In both E1371Q (Fig. 11D) and I344K/ E1371Q (Fig. 11H), SCN- binding had a greater destabilizing impact on Pt(NO2)4 2-binding than did Cl- .
X
ABCC7 p.Ile344Lys 25892339:176:30
status: NEW177 3.6. Effect on single channel conductance The results in Figs. 6 and 7 suggest that antagonistic ion:ion interactions inside the pore that are lost following removal of the positive charge at R334 are at least partially restored by the I344K mutation.
X
ABCC7 p.Ile344Lys 25892339:177:236
status: NEW179 However, the reduced single channel conductance observed in R334Q channels (Fig. 12) was not significantly rescued by the I344K mutation (Fig. 12).
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ABCC7 p.Ile344Lys 25892339:179:122
status: NEW180 This contrasts with the previous observation that the I344K mutation, while having little influence on single channel conductance by itself, could almost completely restore the greatly diminished conductance seen when K95 in the inner vestibule is neutralized by mutagenesis [50].
X
ABCC7 p.Ile344Lys 25892339:180:54
status: NEW182 Fig. 7. Effect of other extracellular anions on Pt(NO2)4 2- block of I344K-containing channels.
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ABCC7 p.Ile344Lys 25892339:182:69
status: NEW183 (A, B) Example macroscopic I-V relationships for I344K/E1371Q (A) or R334Q/I344K/E1371Q (B) with extracellular solution containing 150 mM SCN- .
X
ABCC7 p.Ile344Lys 25892339:183:49
status: NEWX
ABCC7 p.Ile344Lys 25892339:183:75
status: NEW211 Addition of a second positive charge in the inner vestibule in I344K strengthens Pt(NO2)4 2-binding (Figs. 4, 5, 8; Table 1), consistent with this residue also existing close to the internal blocker binding site [35,50] (Fig. 13A).
X
ABCC7 p.Ile344Lys 25892339:211:63
status: NEW216 (A-C) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in E1371Q (A), K95Q/E1371Q (B) and I344K/E1371Q (C) at different membrane potentials as indicated in panel A. Straight-line fits to the data are to Eq. (3) as described in the Materials and methods.
X
ABCC7 p.Ile344Lys 25892339:216:104
status: NEW219 Data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant.
X
ABCC7 p.Ile344Lys 25892339:219:9
status: NEW222 Pt Kvac(0) (bc;M) Pt zb4;vac Pt Kocc(0) (bc;M) Pt zb4;occ Cl K (mM) Cl zb4; E1371Q 245 -0.39 1440 -0.63 179 +0.22 K95Q/E1371Q 1150 -0.23 4400 -0.58 296 +0.20 I344K/E1371Q 0.174 -0.42 978 -1.12 0.264 +1.09 R334Q/E1371Q 1120 -0.71 - - - - R334Q/I344K/E1371Q 31.8 -1.04 1500 -1.15 232 +0.23 Fig. 9. Effect of bound extracellular Cl-ions on the binding of intracellular Pt(NO2)4 2- ions.
X
ABCC7 p.Ile344Lys 25892339:222:173
status: NEWX
ABCC7 p.Ile344Lys 25892339:222:258
status: NEW224 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant.
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ABCC7 p.Ile344Lys 25892339:224:43
status: NEW239 A second mechanism of knock-off in I344K-containing channel pores Addition of a second positive charge lining the inner vestibule in I344K/E1371Q increases intrinsic Pt(NO2)4 2-binding affinity ~1400-fold (Figs. 5, 8; Table 2), likely because the additional positive charge increases electrostatic interactions with divalent Pt(NO2)4 2- anions [47] (Fig. 13B).
X
ABCC7 p.Ile344Lys 25892339:239:35
status: NEWX
ABCC7 p.Ile344Lys 25892339:239:133
status: NEW240 However, this I344K mutation also has a strong influence Fig. 10.
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ABCC7 p.Ile344Lys 25892339:240:14
status: NEW242 (A, B) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in R334Q/E1371Q (A) and R334Q/I344K/E1371Q (B) at different membrane potentials as indicated.
X
ABCC7 p.Ile344Lys 25892339:242:100
status: NEW248 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant.
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ABCC7 p.Ile344Lys 25892339:248:43
status: NEW251 (A, E) Effect of extracellular [SCN- ] ([SCN- ]o) on the measured Pt KD in E1371Q (A) and I344K/E1371Q (E) at 0 mV and -100 mV membrane potential.
X
ABCC7 p.Ile344Lys 25892339:251:90
status: NEW256 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant.
X
ABCC7 p.Ile344Lys 25892339:256:43
status: NEW259 These two effects on binding of internal Pt(NO2)4 2- and external anions suggest that the I344K mutation alters, directly or indirectly, ion interactions with two different binding sites inside the pore.
X
ABCC7 p.Ile344Lys 25892339:259:90
status: NEW260 The alternative to this - that I344K increases the anion binding affinity of a single site that interacts with both internal Pt(NO2)4 2- and external Cl- - can be ruled out based on the shape of the Pt KD-[Cl- ]o relationship for this mutant (Fig. 4J).
X
ABCC7 p.Ile344Lys 25892339:260:31
status: NEW263 Consequently, if we assume that the I344K mutation is increasing the affinity of a Cl-binding site on the extracellular side of the Pt(NO2)4 2-binding site, then this could reflect an increase in the Cl- -binding affinity of the "normal" Cl-binding site in the outer vestibule, or the introduction of a novel Cl-binding site elsewhere in the pore.
X
ABCC7 p.Ile344Lys 25892339:263:36
status: NEW266 However, the R334Q mutation has only a minor impact on Cl-binding in the presence of I344K, and Cl-binding to the R334Q/I344K/E1371Q mutant remains strong and strongly voltage-dependent (Fig. 10; Table 2).
X
ABCC7 p.Ile344Lys 25892339:266:85
status: NEWX
ABCC7 p.Ile344Lys 25892339:266:120
status: NEW267 This suggests that in I344K-containing channels (unlike E1371Q background channels), significant binding of extracellular Cl- occurs at a site that is independent of R334.
X
ABCC7 p.Ile344Lys 25892339:267:22
status: NEW268 Indeed, knock-off of Pt(NO2)4 2- by Cl- appears similar in I344K/ E1371Q and R334Q/I344K/E1371Q channels (Fig. 10D), suggesting that anion binding near R334 plays little role in ion:ion interactions that occur in channels bearing the I344K mutation.
X
ABCC7 p.Ile344Lys 25892339:268:59
status: NEWX
ABCC7 p.Ile344Lys 25892339:268:83
status: NEWX
ABCC7 p.Ile344Lys 25892339:268:234
status: NEW269 Furthermore, the voltage-dependence of external Cl-binding is greatly increased in I344K-containing channels.
X
ABCC7 p.Ile344Lys 25892339:269:83
status: NEW271 The apparent anion selectivity of the external anion binding site is also slightly altered in I344K-containing channels (Fig. 7G, H), and this appears independent of the presence or absence of the R334Q mutation.
X
ABCC7 p.Ile344Lys 25892339:271:94
status: NEW272 Not only the binding affinity of external Cl- and internal Pt(NO2)4 2- , but also the Table 3 Mean parameters obtained from analysis of the [SCN- ]o-dependence of Pt(NO2)4 2- block in E1371Q and I344K/E1371Q, as described in Fig. 11.
X
ABCC7 p.Ile344Lys 25892339:272:195
status: NEW274 Pt Kocc(0) (bc;M) Pt zb4;occ SCN K (bc;M) SCN zb4; E1371Q 17,500 -0.54 14,000 +0.137 I344K/E1371Q 32,900 -0.94 1.95 +0.917 Fig. 12.
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ABCC7 p.Ile344Lys 25892339:274:97
status: NEW276 (A) Example single-channel currents recorded at a membrane potential of -60 mV for the wild type, R334Q, and R334Q/I344K channels as indicated.
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ABCC7 p.Ile344Lys 25892339:276:115
status: NEW282 strengthoftheinteractionbetweenthem,isalteredinI344K-containingchan- nels.Thus,thedestabilizingeffectofCl- onPt(NO2)4 2-binding-evaluated fromthedifferencebetweenPt(NO2)4 2- bindinginvacantandCl- -occupied channels(i.e.betweenPt KvacandPt Kocc)-ismuchgreaterinI344K/E1371Q (and to a lesser extent R334Q/I344K/E1371Q) than in E1371Q or K95Q/ E1371Q(Fig.9).Strengthenedinteractionsbetweenboundanionsarealso suggestedbytheincreasedapparentcouplingbetweenthemovementof Pt(NO2)4 2- andCl- ionsinsidetheporeinI344K(Fig.8E).
X
ABCC7 p.Ile344Lys 25892339:282:303
status: NEW283 One model that appears consistent with these results is that addition of a second positive charge in the inner vestibule (in I344K/E1371Q) increases the number of anions that can bind within this region of the pore (Fig. 13B).
X
ABCC7 p.Ile344Lys 25892339:283:125
status: NEW287 These different mechanisms are illustrated in wild type (A, C) and R334Q/I344K channels (B, D) that are proposed to isolate the two different mechanisms of ion:ion interaction; it is presumed that both mechanisms are active in I344K (and neither in R334Q).
X
ABCC7 p.Ile344Lys 25892339:287:73
status: NEWX
ABCC7 p.Ile344Lys 25892339:287:227
status: NEW289 (B) In R334Q/I344K channels, Cl- does not bind to the outer pore mouth, but instead binds further into the pore from its extracellular end (experiencing a greater fraction of the transmembrane electric field) and binds close to the introduced lysine in the inner vestibule, where it strongly antagonizes Pt(NO2)4 2-binding (leading to very strong [Cl- ]o-dependence of block).
X
ABCC7 p.Ile344Lys 25892339:289:13
status: NEW291 (D) In R334Q/I344K, even if multiple Cl-ions are able to bind close together in the inner vestibule, this does not result in rapid Cl-permeation in the absence of Cl-binding to the outer pore region.
X
ABCC7 p.Ile344Lys 25892339:291:13
status: NEW293 nearby sites could then introduce a novel, strongly destabilizing effect of external Clon internal Pt(NO2)4 2-binding in I344K-containing channels.
X
ABCC7 p.Ile344Lys 25892339:293:121
status: NEW297 In effect, in the R334Q/I344K double pore mutant, one kind of antagonistic Cl- :blocker interaction (Fig. 13A) has been replaced by another with a different molecular mechanism (Fig. 13B).
X
ABCC7 p.Ile344Lys 25892339:297:24
status: NEW298 Presumably in I344K channels both knock-off mechanisms shown in Fig. 13A, B are in effect.
X
ABCC7 p.Ile344Lys 25892339:298:14
status: NEW300 Understanding the observed properties of Pt(NO2)4 2- block of I344K channels Block of I344K/E1371Q by cytoplasmic Pt(NO2)4 2-was previously described as being of very high affinity, very strong voltage dependence, and very high sensitivity to extracellular [Cl- ] [47].
X
ABCC7 p.Ile344Lys 25892339:300:62
status: NEWX
ABCC7 p.Ile344Lys 25892339:300:86
status: NEW311 Reduced conductance in K95Q is restored in K95Q/I344K, leading to the proposal that a positive charge located at either of these two nearby residues is able to support physiologically important Cl-binding in the inner vestibule [35, 50].
X
ABCC7 p.Ile344Lys 25892339:311:48
status: NEW312 However, although I344K appears capable of restoring sensitivity to external Cl-ions that is lost in R334Q, this does not result in a significant restoration of unitary Cl-conductance (Fig. 12).
X
ABCC7 p.Ile344Lys 25892339:312:18
status: NEW313 In other words, although it is suggested that in R334Q/I344K channels one form of knock-off inside the pore has been replaced by another (Fig. 13B), this surrogate knock-off mechanism does not appear to be capable of supporting high Cl-conductance in the way that normal ion-ion interactions in wild type CFTR are proposed to do (Fig. 13C, D).
X
ABCC7 p.Ile344Lys 25892339:313:55
status: NEW