Nau C, Wang S Y, Strichartz G R, Wang G K
Department of Anesthesia Research Laboratories, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Mol Pharmacol. 1999 Aug;56(2):404-13. doi: 10.1124/mol.56.2.404.
Voltage-gated Na(+) channels are the primary targets of local anesthetics (LAs). Amino acid residues in domain 4, transmembrane segment 6 (D4-S6) form part of the LA binding site. LAs inhibit binding of the neurotoxin batrachotoxin (BTX). Parts of the BTX binding site are located in D1-S6 and D4-S6. The affinity of BTX-resistant Na(+) channels mutated in D1-S6 (mu1-N434K, mu1-N437K) toward several LAs is significantly decreased. We have studied how residue mu1-N434 influences LA binding. By using site-directed mutagenesis, we created mutations at mu1-N434 that vary the hydrophobicity, aromaticity, polarity, and charge and investigated their influence on state-dependent binding and stereoselectivity of bupivacaine. Wild-type and mutant channels were transiently expressed in human embryonic kidney 293t cells and investigated under whole-cell voltage-clamp. For resting channels, bupivacaine enantiomers showed a higher potency in all mutant channels compared with wild-type channels. These changes were not well correlated with the physical properties of the substituted residues. Stereoselectivity was small and almost unchanged. In inactivated channels, the potency of bupivacaine was increased in mutations containing a quadrupole of an aromatic group (mu1-N434F, mu1-N434W, mu1-N434Y), a polar group (mu1-N434C), or a negative charge (mu1-N434D) and was decreased in a mutation containing a positive charge (mu1-N434K). In mutation mu1-N434R, containing the positively charged arginine, the potency of S(-)-bupivacaine was selectively decreased, resulting in a stereoselectivity (stereopotency ratio) of 3. Similar results were observed with cocaine but not with RAC 109 enantiomers. We propose that in inactivated channels, residue mu1-N434 interacts directly with the positively charged moiety of LAs and that D1-S6 and D4-S6 form a domain-interface site for binding of BTX and LAs in close proximity.
电压门控钠通道是局部麻醉药（LAs）的主要作用靶点。结构域4跨膜片段6（D4-S6）中的氨基酸残基构成了局部麻醉药结合位点的一部分。局部麻醉药可抑制神经毒素蛙毒素（BTX）的结合。蛙毒素结合位点的部分区域位于D1-S6和D4-S6。在D1-S6中发生突变的抗蛙毒素钠通道（mu1-N434K、mu1-N437K）对几种局部麻醉药的亲和力显著降低。我们研究了残基mu1-N434如何影响局部麻醉药的结合。通过定点诱变，我们在mu1-N434处产生了改变疏水性、芳香性、极性和电荷的突变，并研究了它们对布比卡因状态依赖性结合和立体选择性的影响。野生型和突变型通道在人胚肾293t细胞中瞬时表达，并在全细胞电压钳下进行研究。对于静息通道，与野生型通道相比，布比卡因对映体在所有突变型通道中的效力更高。这些变化与取代残基的物理性质相关性不佳。立体选择性较小且几乎不变。在失活通道中，含有芳香基团（mu1-N434F、mu1-N434W、mu1-N434Y）、极性基团（mu1-N434C）或负电荷（mu1-N434D）的四极子的突变中，布比卡因的效力增加，而在含有正电荷（mu1-N434K）的突变中效力降低。在含有带正电荷精氨酸的突变mu1-N434R中，S(-)-布比卡因的效力选择性降低，导致立体选择性（立体效力比）为3。可卡因也观察到了类似结果，但RAC 109对映体未观察到。我们提出，在失活通道中，残基mu1-N434直接与局部麻醉药的带正电荷部分相互作用，并且D1-S6和D4-S6在近距离形成了一个用于结合蛙毒素和局部麻醉药的结构域界面位点。
