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Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block
Author(s) -
Khan A.,
Romantseva L.,
Lam A.,
Lipkind G.,
Fozzard H. A.
Publication year - 2002
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2002.021014
Subject(s) - chemistry , protonation , conductance , sodium channel , gating , biophysics , xenopus , dissociation constant , stereochemistry , neutralization , patch clamp , skeletal muscle , lysine , voltage clamp , dissociation (chemistry) , proton , crystallography , membrane potential , sodium , amino acid , biochemistry , ion , anatomy , receptor , antibody , biology , gene , immunology , medicine , organic chemistry , combinatorics , quantum mechanics , physics , mathematics
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 p K a (‐log of the dissociation constant) in the acid direction. Effects of neutralization on p K 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 p K a values of carboxylates of Glu‐403, Glu‐758, and Asp‐1532 are shifted to values similar to the experimentally measured p K a . Calculations also predict the experimentally observed changes in p K 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.