Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding

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Abstract
Incubation of purified rat brain sodium channels at 37.degree.C or at high ionic strength causes a concomitant loss of saxitoxin-binding activity and dissociation of .beta.1 subunits. Reaction with hydrophilic carbodiimides produced a resistance against the loss of saxitoxin binding and caused covalent cross-linking of .alpha., 7b1, and .beta.2 subunits. Inthe presence of saxitoxin, this cross-linking reaction led to formation of a state with increased affinity for saxitoxin. However, analysis of the concentration dependence of covalent cross-linking and its inhibition by hydrophilic nucleophiles showed that the stabilization of the saxitoxin-binding activity was due to the formation of a small number of isopeptide bonds in the .alpha. subunit rather than to cross-linking of .alpha. and .beta.1 subunits. In the presence of amine nucleophiles, carbodiimides caused loss of saxitoxin binding, which was prevented in the presence of the toxin. Nucleophiles yielding positiviely charged amide products were more effective than those forming uncharged or negatively products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availablity of carboxyl groups for reaction was increased, prodiving evidence for a toxin-induced conformational change on binding. These results are considered in terms of an allosteric model of saxitoxin binding, in which the functional form of the sodium channel having high affinity for saxitoxin can be stablilized against, inactivation by noncovalent interactions with .beta.1 subunits, binding of saxitoxin and tetrodotoxin, or intramolecular cross-linking of amino acid residues within the .alpha. subunits.