Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage.
Permeation of large tetra-alkylammonium cations through mutant and wild-type voltage-gated sodium channels as revealed by relief of block at high voltage.
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DOI：
10.1085/jgp.115.4.435
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发表时间：
2000-04
期刊：
影响因子：
--
通讯作者：
Moczydlowski E
中科院分区：
文献类型：
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作者：
Huang CJ;Favre I;Moczydlowski E
许多大的有机阳离子是 K+ 通道和属于 P 区超家族的其他阳离子选择性通道的有效阻断剂。然而，大的疏水性阳离子进入和离开这些蛋白质的窄孔的机制尚不清楚。先前的工作表明，电压门控Na+通道DEKA位点中保守的Lys残基是Na+/K+区分、Ca2+排除和有机阳离子分子筛分的重要决定因素。在本研究中，我们试图确定 DEKA 基因座的 Lys(III) 残基是否与内部四烷基铵阳离子 (TAA+) 相互作用，从而以电压依赖性方式阻断 Na+ 通道。我们使用全细胞记录研究了野生型大鼠肌肉 Na+ 通道 (DEKA) 的一系列 TAA+ 阳离子和 DEKA 基因座的两种不同突变体 DEAA 和 DERA 的阻断。 TEA+ 和较大的 TAA+ 阳离子会阻断野生型和 DEAA 通道。然而，DEAA 对大 TAA+ 阳离子的阻滞表现出显着的缓解，如电压大于 +140 mV 时宏观 I-V 曲线的正拐点所揭示的。矛盾的是，在高正电压下观察到大的（例如四戊基铵）而非小（例如 TEA+）对称 TAA+ 阳离子的阻断缓解。 DEKA 野生型通道和 DERA 突变体表现出类似的叠加在背景电流整流上的阻滞现象。结果表明：(a)分子直径达15 Å的疏水性TAA+阳离子在高正电压驱动下可以从内到外渗透Na+通道，(b)DEKA位点的Lys(III)残基是Na+通道内向整流和内部阻断的重要决定因素。根据这些观察结果，我们认为 P 区通道蛋白的亚基、伪亚基或堆积螺旋之间的疏水界面可能有助于促进阻断剂进入孔，因此可能在大 TAA+ 阳离子和潜在的其他类型局麻药分子的阻断和渗透行为中发挥重要作用。
Many large organic cations are potent blockers of K+ channels and other cation-selective channels belonging to the P-region superfamily. However, the mechanism by which large hydrophobic cations enter and exit the narrow pores of these proteins is obscure. Previous work has shown that a conserved Lys residue in the DEKA locus of voltage-gated Na+ channels is an important determinant of Na+/K+ discrimination, exclusion of Ca2+, and molecular sieving of organic cations. In this study, we sought to determine whether the Lys(III) residue of the DEKA locus interacts with internal tetra-alkylammonium cations (TAA+) that block Na+ channels in a voltage-dependent fashion. We investigated block by a series of TAA+ cations of the wild-type rat muscle Na+ channel (DEKA) and two different mutants of the DEKA locus, DEAA and DERA, using whole-cell recording. TEA+ and larger TAA+ cations block both wild-type and DEAA channels. However, DEAA exhibits dramatic relief of block by large TAA+ cations as revealed by a positive inflection in the macroscopic I–V curve at voltages greater than +140 mV. Paradoxically, relief of block at high positive voltage is observed for large (e.g., tetrapentylammonium) but not small (e.g., TEA+) symmetrical TAA+ cations. The DEKA wild-type channel and the DERA mutant exhibit a similar relief-of-block phenomenon superimposed on background current rectification. The results indicate: (a) hydrophobic TAA+ cations with a molecular diameter as large as 15 Å can permeate Na+ channels from inside to outside when driven by high positive voltage, and (b) the Lys(III) residue of the DEKA locus is an important determinant of inward rectification and internal block in Na+ channels. From these observations, we suggest that hydrophobic interfaces between subunits, pseudosubunits, or packed helices of P-region channel proteins may function in facilitating blocker access to the pore, and may thus play an important role in the blocking and permeation behavior of large TAA+ cations and potentially other kinds of local anesthetic molecules.