Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels
Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels
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DOI：
10.1016/s0006-3495(03)74530-5
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发表时间：
2003-08-01
影响因子：
3.4
通讯作者：
Wang, GK
中科院分区：
文献类型：
--
作者：
Wang, SY;Bonner, K;Wang, GK
最近的报道表明，四个S6的C-末端可能共同关闭的电压门控阳离子通道的细胞质侧，可能是一个倒置的圆锥形结构。在这项研究中，我们分别取代的D1 S6和D4 S6的C-末端的色氨酸（W）的rNav1.4 Na+通道α-亚基的总共18个残基，并检查其相应的门控特性时，在Hek 293 t细胞沿着与β 1亚基表达。几种W突变体在激活、快速失活和/或缓慢失活门控中显示出显著变化。特别是，5个S6 W-突变体表现出不完全的快速失活与非失活维持电流存在。这五个残基的半胱氨酸（C）取代导致两个突变体具有稍微更多的维持电流。这五个位置的多个取代产生了两个突变体（L437 C/A438 W、L435 W/L437 C/A438 W），其表现出最小快速失活的表型。出乎意料的是，这种失活缺陷突变体表达Na+电流以及野生型。此外，所有突变体与受损的快速失活表现出增强的慢失活表型。这些结果的影响将讨论通过氨基酸取代和/或直接参与钠通道门控的S6 C-末端的间接变构调制。
Recent reports suggest that four S6 C-termini may jointly close the voltage-gated cation channel at the cytoplasmic side, probably as an inverted teepee structure. In this study we substituted individually a total of 18 residues at D1S6 and D4S6 C-terminal ends of the rNav1.4 Na+ channel alpha-subunit with tryptophan (W) and examined their corresponding gating properties when expressed in Hek293t cells along with beta1 subunit. Several W-mutants displayed significant changes in activation, fast inactivation, and/or slow inactivation gating. In particular, five S6 W-mutants showed incomplete fast inactivation with noninactivating maintained currents present. Cysteine (C) substitutions of these five residues resulted in two mutants with slightly more maintained currents. Multiple substitutions at these five positions yielded two mutants (L437C/A438W, L435W/L437C/A438W) that exhibited phenotypes with minimal fast inactivation. Unexpectedly, such inactivation-deficient mutants expressed Na+ currents as well as did the wild-type. Furthermore, all mutants with impaired fast inactivation exhibited an enhanced slow inactivation phenotype. Implications of these results will be discussed in terms of indirect allosteric modulations via amino acid substitutions and/or a direct involvement of S6 C-termini in Na+ channel gating.