Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: Kinetic analysis of mutual inhibition between μ-conotoxin GIIIA derivatives and amine blockers

Quanli Ma, Evgeny Pavlov, Tatiana Britvina, Gerald W. Zamponi, Robert J. French

Research output: Contribution to journalArticle

Abstract

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Nachannels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA],based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +150 mV, for TPrA (∼4mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

Original languageEnglish (US)
Pages (from-to)4266-4276
Number of pages11
JournalBiophysical Journal
Volume95
Issue number9
DOIs
StatePublished - Nov 1 2008

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Batrachotoxins
Conotoxins
Sodium Channels
Amines
Skeletal Muscle
Static Electricity
Membranes

ASJC Scopus subject areas

  • Biophysics

Cite this

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels : Kinetic analysis of mutual inhibition between μ-conotoxin GIIIA derivatives and amine blockers. / Ma, Quanli; Pavlov, Evgeny; Britvina, Tatiana; Zamponi, Gerald W.; French, Robert J.

In: Biophysical Journal, Vol. 95, No. 9, 01.11.2008, p. 4266-4276.

Research output: Contribution to journalArticle

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AB - R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean "blocked" durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Nachannels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude ("fast" block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA],based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +150 mV, for TPrA (∼4mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.

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