ABCMdb

ABCC7 p.Val1056His

Predicted by SNAP2:	A: N (82%), C: N (78%), D: D (71%), E: N (53%), F: N (66%), G: N (61%), H: D (75%), I: N (93%), K: N (53%), L: N (72%), M: N (66%), N: D (59%), P: N (53%), Q: N (66%), R: N (57%), S: N (61%), T: N (82%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: N, Y: N,

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Publications
[hide] CFTR: effect of ICL2 and ICL4 amino acids in close... J Cyst Fibros. 2013 Dec;12(6):737-45. doi: 10.1016/j.jcf.2013.02.002. Epub 2013 Mar 9. Billet A, Mornon JP, Jollivet M, Lehn P, Callebaut I, Becq F
CFTR: effect of ICL2 and ICL4 amino acids in close spatial proximity on the current properties of the channel.
J Cyst Fibros. 2013 Dec;12(6):737-45. doi: 10.1016/j.jcf.2013.02.002. Epub 2013 Mar 9., [PMID:23478129]

Abstract [show]
BACKGROUND: CFTR is the only ABC transporter functioning as a chloride (Cl(-)) channel. We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. METHODS: Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. RESULTS: We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl(-) current of CFTR (couple E267-K1060) or ii) increases it with a change of the electrophysiological signature (couple S263-V1056). CONCLUSIONS: These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl(-) permeation pathway of CFTR.

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Sentences [show]
No. Sentence Comment
113 To investigate the influence of these two amino acids on CFTR channel function, we substituted one or both residues by a histidine residue, which is a potentially positively charged residue as it is protonable in a pH-dependent manner (pKa of ~6), creating thereby the mutants S263H- and V1056H- as well as the double-mutant S263H-V1056H-CFTR. Login to comment
116 Importantly upon addition of forskolin, the electrophysiological properties of the ICL2 S263H and ICL4 V1056H mutants were clearly different from those observed with wt CFTR: the CFTR current observed with both mutants was time and voltage dependent (Fig. 3C-D). Login to comment
117 At positive potentials, the outward current of S263H and V1056H mutants greatly increases compared to wt, but contrary to wt CFTR currents, it deactivates during the pulse potential (Figs. 3C-D, 4). Login to comment
120 Interestingly, in the case of the double mutant S263H-V1056H, we observed the restoration of the time and voltage independence of the current as well as of the level of the current density (Fig. 3C-D, Table 1). Login to comment
143 -100 -50 100 50 C D A S263H-V1056H S263H V1056H I (pA /pF) V (mV) S263H V1056H S263H-V1056H 5000pA 50ms B 600 400 200 -200 Fig. 3. Login to comment
150 (C, D) Whole cell chloride current of HEK293 cells expressing S263H-CFTR, V1056H-CFTR or the double mutant S263H-V1056H-CFTR obtained in presence of 10 bc;M Fsk. Login to comment
174 I (pA/pF) at -100 mV Basal Fsk/start of the pulse Fsk/end of the pulse +CFTRinh172 Mean SEM N Mean SEM N t test Mean SEM N t test Mean SEM N wt -3.4 0.9 18 -100.4 17.5 18 -74.3 7.3 18 -11.2 3.0 18 S263H -3.1 0.8 8 -207.0 37.8 8 ** -84.6 13.9 8 ns -5.9 1.0 8 V1056H -2.0 0.6 7 -213.0 31.6 7 ** -95.0 18.7 7 ns -4.1 0.9 7 S263H-V1056H -1.8 0.4 16 -96.0 14.5 16 ns -80.5 9.3 16 ns -7.9 1.0 16 I (pA/pF) at +100 mV Basal Fsk/start of the pulse Fsk/end of the pulse +CFTRinh172 Mean SEM N Mean SEM N t test Mean SEM N t test Mean SEM N wt 14.5 4.1 18 260.5 46.1 18 233.6 25.0 18 35.9 6.2 18 S263H 13.1 3.5 8 573.5 75.5 8 ** 523.7 60.5 8 *** 25.8 6.7 8 V1056H 5.7 1.9 7 588.8 68.6 7 *** 531.9 65.6 7 *** 14.9 4.2 7 S263H-V1056H 7.8 1.2 16 279.1 40.5 16 ns 264.7 30.7 16 ns 14.0 4.8 16 ns: not significant difference; **: pb0.01; ***: pb0.001. Login to comment
177 Clearly, the understanding of the reasons underlying the outward rectification seen in both S263H and V1056H mutants requires further in-depth investigations, nevertheless the effects of those mutations on current density and voltage-dependence suggest that this region of the protein may play a critical role in the Cl- pathway and may be physically close to the inner mouth of the pore. Login to comment
178 Restoration of the voltage independence and of a current density similar to wt in the double mutant S263H-V1056H might be explained by the probable tight contact between the two histidine residues, similarly to the situation occurring in the Sav1866 template, a contact possibly counteracting the effects of the single mutant (S263H or V1056H) on current density. Login to comment