Linford N J, Cantrell A R, Qu Y, Scheuer T, Catterall W A
Department of Pharmacology, Box 357280, University of Washington, Seattle, WA 98195-7280, USA.
Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13947-52. doi: 10.1073/pnas.95.23.13947.
The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-alpha-benzoate binding affinity, causing a 60-fold increase in Kd. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in Kd. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation approximately 40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to approximately 60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain-interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on L-type calcium channels.
电压门控钠通道是六类以上神经毒素和药物的作用位点，这些毒素和药物通过与不同的、变构偶联的受体位点相互作用来改变其功能。蟾毒素（BTX）是一种甾体生物碱，它与神经毒素受体位点2结合并导致持续激活。BTX的结合受到局部麻醉剂的变构抑制。我们研究了BTX与氨基酸残基I1760、F1764和Y1771的相互作用，这些残基构成了IIA型钠通道跨膜片段IVS6中局部麻醉剂受体位点的一部分。用丙氨酸替代F1764（突变体F1764A）会降低氚化BTX-A-20-α-苯甲酸酯的结合亲和力，导致解离常数（Kd）增加60倍。在预测的IVS6跨膜α螺旋中与F1764相邻的I1760用丙氨酸替代，只会使Kd增加4倍。相比之下，突变体Y1771A的BTX结合亲和力没有变化。对于野生型和突变体Y1771A，BTX使半数最大激活电压在超极化方向上大约偏移40 mV，并使非失活钠电流的百分比增加到大约60%。相比之下，这些BTX效应在F1764A突变体中完全消除，在突变体I1760A中则大幅降低。我们的数据表明，BTX受体位点与跨膜片段IVS6中的局部麻醉剂受体位点共享重叠但不完全相同的分子决定因素，并且如先前工作所示，在跨膜片段IS6中具有独特的分子决定因素。显然，BTX符合先前针对作用于L型钙通道的钙激动剂和拮抗剂药物提出的配体结合和作用的结构域-界面变构模型。
