Wang G K, Quan C, Wang S Y
Department of Anesthesia, Harvard Medical School and Brigham & Women's Hospital, Boston, Massachusetts 02115, USA.
Mol Pharmacol. 1998 Aug;54(2):389-96. doi: 10.1124/mol.54.2.389.
Local anesthetics (LAs) are noncompetitive antagonists of batrachotoxin (BTX) in voltage-gated Na+ channels. The putative LA receptor has been delineated within the transmembrane segment S6 in domain IV of voltage-gated Na+ channels, whereas the putative BTX receptor is within segment S6 in domain I. In this study, we created BTX-resistant muscle Na+ channels at segment I-S6 (micro1-N434K, micro1-L437K) to test whether these residues modulate LA binding. These mutant channels were expressed in transiently transfected human embryonic kidney 293T cells, and their sensitivity to lidocaine, QX-314, etidocaine, and benzocaine was assayed under whole-cell, voltage-clamp conditions. Our results show that LA binding in BTX-resistant micro1 Na+ channels was reduced significantly. At -100 mV holding potential, the reduction in LA affinity was maximal for QX-314 (by 17-fold) and much less for neutral benzocaine (by 2-fold). Furthermore, this reduction was residue specific; substitution of positively charged lysine with negatively charged aspartic acid (micro1-N434D) restored or even enhanced the LA affinity. We conclude that micro1-N434K and micro1-L437K residues located near the middle of the I-S6 segment of Na+ channels can reduce the LA binding affinity without BTX. Thus, this reduction of the LA affinity by point mutations at the BTX binding site is not caused by gating changes induced by BTX alone. We surmise that the BTX receptor and the LA receptor within segments I-S6 and IV-S6, respectively, may align near or within the Na+ permeation pathway.
局部麻醉药（LAs）是电压门控Na⁺通道中蟾毒素（BTX）的非竞争性拮抗剂。推测的局部麻醉药受体已在电压门控Na⁺通道结构域IV的跨膜片段S6内确定，而推测的BTX受体在结构域I的片段S6内。在本研究中，我们在片段I-S6处创建了对BTX耐药的肌肉Na⁺通道（micro1-N434K、micro1-L437K），以测试这些残基是否调节局部麻醉药的结合。这些突变通道在瞬时转染的人胚肾293T细胞中表达，并在全细胞电压钳条件下测定它们对利多卡因、QX-314、依替卡因和苯佐卡因的敏感性。我们的结果表明，对BTX耐药的micro1 Na⁺通道中局部麻醉药的结合显著减少。在-100 mV的钳制电位下，QX-314的局部麻醉药亲和力降低最大（降低17倍），而中性苯佐卡因的降低则少得多（降低2倍）。此外，这种降低是残基特异性的；用带负电荷的天冬氨酸替代带正电荷的赖氨酸（micro1-N434D）可恢复甚至增强局部麻醉药的亲和力。我们得出结论，位于Na⁺通道I-S6片段中部附近的micro1-N434K和micro1-L437K残基可在无BTX的情况下降低局部麻醉药的结合亲和力。因此，BTX结合位点处的点突变导致的局部麻醉药亲和力降低并非仅由BTX诱导的门控变化引起。我们推测，分别位于片段I-S6和IV-S6内的BTX受体和局部麻醉药受体可能在Na⁺通透途径附近或之内排列。
