Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block.
Voltage-gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure-function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Na(v)1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two-micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half-activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pK(a) (-log of the dissociation constant) in the acid direction. Effects of neutralization on pK(a) were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pK(a) values of carboxylates of Glu-403, Glu-758, and Asp-1532 are shifted to values similar to the experimentally measured pK(a). Calculations also predict the experimentally observed changes in pK(a) that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.
The Cardiac Electrophysiology Laboratories, Department of Medicine, The University of Chicago, Chicago, IL 60637, USA.
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Khan
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Romantseva
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Lam
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Lipkind
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Fozzard
D000818:Animals; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D016296:Mutagenesis / Q000502:physiology; D009865:Oocytes / Q000502:physiology; D020836:Protein Structure, Quaternary; D011522:Protons; D051381:Rats; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D014981:Xenopus
D011522:Protons; D015222:Sodium Channels; D012964:Sodium