ABCC7 p.Val345His
| 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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No. Sentence Comment
78 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).
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ABCC7 p.Val345His 23083715:78:44
status: NEWX
ABCC7 p.Val345His 23083715:78:150
status: NEW80 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).
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ABCC7 p.Val345His 23083715:80:111
status: NEW81 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).
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ABCC7 p.Val345His 23083715:81:27
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.Val345His 23083715:82:152
status: NEW102 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.
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ABCC7 p.Val345His 23083715:102:125
status: NEW103 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.
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ABCC7 p.Val345His 23083715:103:178
status: NEW128 (A) Example leak-subtracted macroscopic I/V relationships for V345H at intracellular pH of 5.5 (left) or 9.0 (right, different patch).
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ABCC7 p.Val345His 23083715:128:62
status: NEW130 (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.
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ABCC7 p.Val345His 23083715:130:87
status: NEW138 (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).
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ABCC7 p.Val345His 23083715:138:47
status: NEW140 (B) Mean single-channel i/V relationships for WT and V345H under these two pH conditions.
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ABCC7 p.Val345His 23083715:140:53
status: NEW148 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).
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ABCC7 p.Val345His 23083715:148:162
status: NEW