Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, 100122, China.
Department of Pharmaceutical Toxicology, School of Pharmacy, China Medical University, Shenyang, 100122, China.
Biochem Biophys Res Commun. 2022 Oct 30;627:39-44. doi: 10.1016/j.bbrc.2022.07.056. Epub 2022 Jul 19.
Na1.5 channel is an integral membrane protein involved in the initiation and conduction of action potentials. IQ motif is located in the C-terminal domain of Na1.5 sodium channel, which is highly conserved in human sodium channel subtypes. IQ motif is involved in the Ca-dependent regulation through interaction with the regulatory proteins such as calpastatin domain L (CS). Mutations in SCN5A, the gene encoding Na1.5 channel, have been linked to many cardiac arrhythmias, such as Long QT syndrome type 3 (LQT3) and Brugada syndrome (BRS). LQT3-associated mutations in Na1.5 IQ motif, IQ and IQ, have been reported to affect the late I. A BRS-associated mutation in Na1.5 IQ motif, IQ has been reported to affect the peak I. But the detailed pathogenic mechanisms of LQT3 and BRS remains unclear. To explore the binding properties of CS to IQ motif and its muants associated with LQT3/BRS, molecular docking and GST pull down assay were performed in this study. As a result, S58 and E59 in CS activating channel effect region L54-64 were involved in the conformation of the CS/IQ complex by protein-protein docking. IQ motif could bind to CS in a [CS]-dependent and [Ca]-dependent manner by pull down assay. However, the binding affinities of IQ and IQ to CS were decreased and its reaction rates with CS were slower. The binding characteristics of IQ to CS was opposite in a [Ca]-dependent manner and its binding efficacy became smaller. The changes of the binding characteristics of IQ to CS would affect the regulation of Na1.5 channel, which may be related to LQT3 and BRS.
钠通道 1.5 型（Na1.5）是一种整合膜蛋白，参与动作电位的启动和传导。IQ 基序位于 Na1.5 钠通道的 C 端结构域，在人类钠通道亚型中高度保守。IQ 基序通过与钙调蛋白依赖性蛋白酶抑制剂（calpastatin domain L，CS）等调节蛋白相互作用，参与 Ca2+依赖性调节。编码 Na1.5 通道的 SCN5A 基因突变与多种心律失常有关，如长 QT 综合征 3 型（LQT3）和 Brugada 综合征（BRS）。已有研究报道 Na1.5 IQ 基序、IQ 和 IQ 相关的 LQT3 突变会影响晚期 I。BRS 相关的 Na1.5 IQ 基序、IQ 突变会影响峰值 I。但 LQT3 和 BRS 的详细发病机制仍不清楚。为了探讨 CS 与 LQT3/BRS 相关的 IQ 基序及其突变体的结合特性，本研究进行了分子对接和 GST 下拉实验。结果表明，CS 激活通道效应区 L54-64 的 S58 和 E59 通过蛋白-蛋白对接参与 CS/IQ 复合物的构象。通过下拉实验发现 IQ 基序可与 CS 以 [CS]-依赖性和 [Ca2+]-依赖性方式结合。然而，IQ 和 IQ 与 CS 的结合亲和力降低，与 CS 的反应速率变慢。IQ 与 CS 的结合特性呈相反的 [Ca2+]-依赖性变化，其结合效力变小。IQ 与 CS 结合特性的变化会影响 Na1.5 通道的调节，可能与 LQT3 和 BRS 有关。
