Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding.
Incubation of purified rat brain sodium channels at 37 degrees 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, beta 1, and beta 2 subunits. In the 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 positively charged amide products were more effective than those forming uncharged or negatively charged products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availability of carboxyl groups for reaction was increased, providing 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 stabilized 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 subunit.
Tejedor
F J
FJ
Department of Pharmacology, University of Washington, Seattle 98195.
McHugh
E
E
Catterall
W A
WA
D000494:Allosteric Regulation; D029845:Amphibian Proteins; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D002234:Carbodiimides / Q000494:pharmacology; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009994:Osmolar Concentration; D051381:Rats; D012530:Saxitoxin / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
D029845:Amphibian Proteins; D002234:Carbodiimides; D002352:Carrier Proteins; D004220:Disulfides; D007473:Ion Channels; D046911:Macromolecular Substances; C027525:saxitoxin-binding protein, Rana catesbeiana; C026512:arginine methyl ester; D012530:Saxitoxin; D001120:Arginine; D012964:Sodium; D004229:Dithiothreitol