Lampert, A. and O’Reilly, Andrias O. and Dib-Hajj, S.D. and Tyrrell, L. and Wallace, Bonnie A. and Waxman, S.G. (2008) A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Na(v)17 channel is disrupted by the erythromelalgia-associated F1449V mutation. Journal of Biological Chemistry 283 , pp. 24118-24127. ISSN 0021-9258.
Abstract
Sodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7.
Metadata
| Item Type: | Article |
|---|---|
| School: | Birkbeck Faculties and Schools > Faculty of Science > School of Natural Sciences |
| Research Centres and Institutes: | Bioinformatics, Bloomsbury Centre for (Closed), Structural Molecular Biology, Institute of (ISMB) |
| Depositing User: | Administrator |
| Date Deposited: | 04 Aug 2010 14:09 |
| Last Modified: | 02 Aug 2023 16:49 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/1183 |
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