Point mutations at L1280 in Nav1.4 channel D3-S6 modulate binding affinity and stereoselectivity of bupivacaine enantiomers
Point mutations at L1280 in Nav1.4 channel D3-S6 modulate binding affinity and stereoselectivity of bupivacaine enantiomers
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DOI：
10.1124/mol.63.6.1398
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发表时间：
2003-06-01
影响因子：
3.6
通讯作者：
Wang, GK
中科院分区：
文献类型：
--
作者：
Nau, C;Wang, SY;Wang, GK
局部麻醉剂 (LA) 阻断电压门控钠通道。 LA 结合位点的部分位于结构域 1、3 和 4 的孔内衬跨膜片段 6（D1-S6、D3-S6、D4-S6）。我们之前提出，D1-S6 中的残基 N434 直接与失活通道中的布比卡因对映体相互作用，因为 N434 处取代的不同残基的侧链特性与布比卡因对映体阻断效力的变化相关。此外，突变N434R对失活通道的阻断表现出显着的立体选择性，这是由于S(-)-布比卡因的阻断选择性减少所致。在本研究中，我们分析了大鼠骨骼肌Na(v)1.4通道D3-S6中残基L1280在与布比卡因对映体相互作用中的作用。我们用不同理化性质的氨基酸取代了 L1280 处的天然亮氨酸。野生型和突变型通道在人胚胎肾 293t 细胞中瞬时表达，并在全细胞电压钳下进行研究。布比卡因对映体对静息突变体通道的阻断显示与野生型通道相比几乎没有差异。含有芳香族基团的突变（L1280W）中失活通道的阻断增加，而含有正电荷的突变（L1280K、L1280R）中失活通道的阻断减少。令人惊讶的是，突变体L1280E、L1280N、L1280Q和L1280R对失活通道的阻断表现出显着的立体选择性。更令人惊讶的是，与D1-S6中的突变N434R相比，立体选择性是由R(+)-布比卡因阻断的选择性减少引起的。我们提出，在失活的通道中，D3-S6 中的残基 L1280 和 D1-S6 中的 N434 残基直接与 LA 相互作用，从而在离子传导孔中彼此面对。
Local anesthetics (LAs) block voltage-gated sodium channels. Parts of the LA binding site are located in the pore-lining transmembrane segments 6 of domains 1, 3, and 4 ( D1-S6, D3-S6, D4-S6). We suggested previously that residue N434 in D1-S6 interacts directly with bupivacaine enantiomers in inactivated channels because side-chain properties of different residues substituted at N434 correlated with changes in blocking potencies of bupivacaine enantiomers. Furthermore, mutation N434R exhibited significant stereoselectivity for block of inactivated channels that resulted from a selective decrease in block by S(-)- bupivacaine. In the present study, we analyzed the role of residue L1280 in D3-S6 of the rat skeletal muscle Na(v)1.4 channel in interactions with the enantiomers of bupivacaine. We substituted native leucine at L1280 with amino acids of different physicochemical properties. Wild-type and mutant channels were expressed transiently in human embryonic kidney 293t cells and were investigated under whole-cell voltage clamp. Block of resting mutant channels by bupivacaine enantiomers revealed little difference compared with wild-type channels. Block of inactivated channels was increased in a mutation containing an aromatic group ( L1280W) and decreased in mutations containing a positive charge ( L1280K, L1280R). Surprisingly, mutants L1280E, L1280N, L1280Q, and L1280R exhibited significant stereoselectivity for block of inactivated channels. More surprisingly, stereoselectivity resulted from a selective decrease in block by R(+)-bupivacaine, in contrast to mutation N434R in D1-S6. We propose that in inactivated channels, residues L1280 in D3-S6 and N434 in D1-S6 interact directly with LAs and thereby face each other in the ion-conducting pore.