Disparate role of Na(+) channel D2-S6 residues in batrachotoxin and local anesthetic action.
Disparate role of Na(+) channel D2-S6 residues in batrachotoxin and local anesthetic action.
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DOI：
10.1124/mol.59.5.1100
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发表时间：
2001-05
影响因子：
3.6
通讯作者：
Sho‐Ya Wang;Maria Barile;G. Wang
中科院分区：
文献类型：
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作者：
Sho‐Ya Wang;Maria Barile;G. Wang
蝙蝠毒素(BTX)稳定电压门控Na(+)通道的开放构象，而局麻药(LAS)则阻断Na(+)电导。定点突变已经确定了在阿尔法亚单位Na(+)通道中的D1-S6、D3-S6和D4-S6片段上的共同残基簇，这些残基对这两种类型的配体的结合至关重要。在本报告中，我们讨论D2-S6部门是否同样参与了BTX和LA的行动。大鼠骨骼肌钠离子通道((MU)1/Skm1)的G783~L795位13个氨基酸分别被赖氨酸残基取代。4个突变体(N784K、L785K、V787K和L788K)表达了足够的Na(+)电流，为进一步研究奠定了基础。突变通道的激活和/或失活门控发生改变；尤其是，MU1-V787K表现出增强的缓慢失活，并在重复脉冲期间表现出对峰Na(+)电流的使用依赖性抑制。其中两个突变体(U)1-N784K和(U)1-L788K对5微米BTX具有完全抗性。这种BTX抗性表型可能是由D2-S6片段的赖氨酸点突变引起的结构扰动引起的。然而，这两个BTX抗性突变体对布比卡因阻断仍然非常敏感，对灭活的Na(+)通道(K(I))的亲和力约为10微米或更小，这表明(Mu)1-N784和(Mu)1-L788残基不太靠近LA结合部位。
Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.