Critical molecular determinants of voltage-gated sodium channel sensitivity to μ-conotoxins GIIIA/B
Critical molecular determinants of voltage-gated sodium channel sensitivity to μ-conotoxins GIIIA/B
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DOI：
10.1124/mol.61.5.1192
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发表时间：
2002-05-01
影响因子：
3.6
通讯作者：
Dib-Hajj, SD
中科院分区：
文献类型：
--
作者：
Cummins, TR;Aglieco, F;Dib-Hajj, SD
GIIIA/B μ-芋螺毒素通过与孔中的特定残基结合以高亲和力阻断大鼠骨骼肌钠通道（rNa（v）1.4）。然而，对GIIIA/B阻断有抗性的人Na（v）1.4（hNa（v）1.4）通道具有这些相同的孔残基。我们使用嵌合体结构，定点诱变，和电生理技术，以调查哪些残基决定GIIIA/B的选择性。大鼠Na（v）1.4的D2/S5-S6接头中的丝氨酸729与hNa（v）1.4中的相应残基亮氨酸（S729 L）的交换使rNa（v）1.4的敏感性降低了约20倍，并且在很大程度上解释了rNa（v）1.4和hNa（v）1.4对GIIIA和GIIIB两者的不同敏感性。为了确定D2/S5-S6接头残基是否可能有助于神经元通道对GIIIA/B的抗性，我们交换了该接头中在rNa（v）1.4和神经元通道之间不同的残基。用赖氨酸（N732 K）取代精氨酸732（rNa（v）1.1a和rNa（v）1.7中的相应残基），可使rNa（v）1.4的GIIIB敏感性降低约20倍。然而，N732 K取代仅使rNa（v）1.4的GIIIA敏感性降低了约4倍，表明GIIIA和GIIIB与D2/S5-S6接头具有不同的相互作用。我们的数据表明，在D2/S5-S6接头的孔外区域中天然存在的变体有助于钠通道对GIIIA/B的亚型特异性敏感性。由于S729和N732不是μ-芋螺毒素的高亲和力结合位点的一部分，这些孔外残基可能影响毒素对孔内结合位点的可及性和/或毒素-通道复合物的稳定性。我们的研究结果应该有助于开发阻断特定神经元钠通道亚型的毒素。
GIIIA/B mu-conotoxins block the rat skeletal muscle sodium channel (rNa(v) 1.4) with high affinity by binding to specific residues in the pore. However, human Na(v)1.4 (hNa(v)1.4) channels, which are resistant to block by GIIIA/B, have these same pore residues. We used chimera constructs, site-directed mutagenesis, and electrophysiological techniques to investigate which residues determine GIIIA/B selectivity. Exchange of serine 729 in the D2/S5-S6 linker of rat Na(v)1.4 with leucine (S729L), the corresponding residue in hNa(v)1.4, reduces the sensitivity of rNa(v)1.4 by similar to20-fold and largely accounts for the differential sensitivity of rNa(v)1.4 and hNa(v)1.4 to both GIIIA and GIIIB. To determine whether D2/S5-S6 linker residues might contribute to the resistance of neuronal channels to GIIIA/B, we exchanged residues in this linker that differed between rNa(v)1.4 and neuronal channels. Substitution of aspargine 732 with lysine (N732K), the corresponding residue in rNa(v)1.1a and rNa(v)1.7, reduced the GIIIB sensitivity of rNa(v)1.4 by similar to20-fold. The N732K substitution, however, only reduced GIIIA sensitivity of rNa(v)1.4 by similar to4-fold, demonstrating that GIIIA and GIIIB have distinct interactions with the D2/S5-S6 linker. Our data indicate that naturally occurring variants in the extra-pore region of the D2/S5-S6 linker contribute to the isoform-specific sensitivity of sodium channels to GIIIA/B. Because S729 and N732 are not part of the high-affinity binding site for mu-conotoxins, these extra-pore residues probably influence the accessibility of the toxin to the binding site within the pore and/or the stability of the toxin-channel complex. Our results should aid the development of toxins that block specific neuronal sodium channel isoforms.