ABCC8 p.Cys418Ala
| Predicted by SNAP2: | A: D (75%), D: D (75%), E: D (95%), F: D (59%), G: D (66%), H: D (63%), I: N (61%), K: D (66%), L: D (53%), M: N (53%), N: N (53%), P: D (80%), Q: D (53%), R: D (95%), S: N (57%), T: N (72%), V: N (82%), W: D (85%), Y: D (66%), |
| Predicted by PROVEAN: | A: N, D: N, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N, |
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[hide] Mutations at lipid-exposed residues of the acetylc... Mol Pharmacol. 1998 Jul;54(1):146-53. Bouzat C, Roccamo AM, Garbus I, Barrantes FJ
Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.
Mol Pharmacol. 1998 Jul;54(1):146-53., [PMID:9658200]
Abstract [show]
The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.
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No. Sentence Comment
103 A minor component of 1.03 Ϯ 0.16 msec (relative area, 0.20 Ϯ 0.03) was observed in 40% of the recordings from C418A-containing channels.
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ABCC8 p.Cys418Ala 9658200:103:122
status: NEW105 ␣ Subunit on burst n/b Current n msec pA Adult (-containing) AChR Wild-type 0.98 Ϯ 0.23 1.25 Ϯ 0.19 1.08 Ϯ 0.10 5.70 Ϯ 0.30 6 M4ϫ4 0.23 Ϯ 0.02a 0.29 Ϯ 0.10a 1.09 Ϯ 0.05 5.50 Ϯ 0.20 6 L411A 0.93 Ϯ 0.16 1.06 Ϯ 0.15 1.12 Ϯ 0.10 5.60 Ϯ 0.10 5 M415A 1.30 Ϯ 0.25 1.49 Ϯ 0.21 1.10 Ϯ 0.02 5.80 Ϯ 0.12 3 C418A 0.31 Ϯ 0.07a 0.36 Ϯ 0.07a 1.03 Ϯ 0.02 5.80 Ϯ 0.10 5 T422A 0.18 Ϯ 0.03a 0.20 Ϯ 0.04a 1.04 Ϯ 0.03 5.70 Ϯ 0.24 6 F414A 1.34 Ϯ 0.30 1.49 Ϯ 0.40 1.08 Ϯ 0.06 5.80 Ϯ 0.25 8 Embryonic (␥-containing) AChR Wild-type 5.51 Ϯ 0.51 9.20 Ϯ 1.41 1.70 Ϯ 0.14 3.85 Ϯ 0.13 6 C418A 2.82 Ϯ 0.56a 4.03 Ϯ 0.90a 1.28 Ϯ 0.10 3.67 Ϯ 0.14 6 T422A 1.55 Ϯ 0.03a 1.80 Ϯ 0.05a 1.20 Ϯ 0.10 4.00 Ϯ 0.20 6 a Statistically different from the wild-type value (p Ͻ 0.001).
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ABCC8 p.Cys418Ala 9658200:105:428
status: NEW126 When T422A or C418A mutant ␣ Fig. 2.
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ABCC8 p.Cys418Ala 9658200:126:14
status: NEW147 To determine whether the mutations in ␣M4 introduced changes in equilibrium agonist binding, we compared the inhibition of ␣-BTX binding by the agonist CCh in the two ␣M4 mutants, C418A, and T422A.
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ABCC8 p.Cys418Ala 9658200:147:201
status: NEW154 The largest decrease was observed with the C418A mutant (Fig. 6).
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ABCC8 p.Cys418Ala 9658200:154:43
status: NEW216 The curves are fits to the Hill equation with the following parameters: ␣2beta⑀␦: Kd ϭ 2 ϫ 10-5 M, nH ϭ 1.18; ␣2(C418A)beta⑀␦: Kd ϭ 2 ϫ 10-5 M, nH ϭ 1.10; ␣2(T422A)beta⑀␦: Kd ϭ 6 ϫ 10-5 M, nH ϭ 1.23.
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ABCC8 p.Cys418Ala 9658200:216:159
status: NEW