Tejedor F J, McHugh E, Catterall W A
Department of Pharmacology, University of Washington, Seattle 98195.
Biochemistry. 1988 Apr 5;27(7):2389-97. doi: 10.1021/bi00407a021.
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.
将纯化的大鼠脑钠通道在37℃或高离子强度下孵育会导致石房蛤毒素结合活性随之丧失以及β1亚基解离。与亲水性碳二亚胺反应产生了对石房蛤毒素结合丧失的抗性，并导致α、β1和β2亚基的共价交联。在石房蛤毒素存在的情况下，这种交联反应导致形成了对石房蛤毒素亲和力增加的状态。然而，对共价交联的浓度依赖性及其被亲水性亲核试剂抑制的分析表明，石房蛤毒素结合活性的稳定是由于在α亚基中形成了少量异肽键，而不是α和β1亚基的交联。在胺亲核试剂存在的情况下，碳二亚胺导致石房蛤毒素结合丧失，而在毒素存在时这种情况得以防止。产生带正电荷酰胺产物的亲核试剂比形成不带电荷或带负电荷产物的亲核试剂更有效。在石房蛤毒素保护钠通道结合活性免于失活的条件下，用于反应的羧基的总体可用性增加，这为毒素结合时诱导的构象变化提供了证据。根据石房蛤毒素结合的变构模型来考虑这些结果，在该模型中，对石房蛤毒素具有高亲和力的钠通道功能形式可以通过与β1亚基的非共价相互作用、石房蛤毒素和河豚毒素的结合或α亚基内氨基酸残基的分子内交联而稳定免于失活。
