鈣調素對電壓依賴型鈉離子通道的調控之研究

Abstract

電壓依賴型鈉離子通道(voltage-gated sodium channel, VGSCs)在引起細胞動作電位(action potential)及動作電位的傳遞上扮演重要角色。鈣調素(calmodulin)是一胞內鈣離子感受蛋白，可透過膜通道蛋白C端上高度保守性之蛋白序列IQ motif對許多不同類型之膜通道蛋白進行調控。前人的研究當中指出，鈣調素對於不同的類型鈉離子通道蛋白之IQ motif具有不同親和能力。本實驗當中，將鈣調素(CaM)以及失去正常鈣離子結合功能之突變型鈣調素(CaM1234)與電壓依賴型鈉離子通道(Nav1.4)同時表現於人類胚胎腎臟細胞(human embryonic kidney cells, HEK 293T)，鈣調素(CaM)和突變型鈣調素(CaM1234)可增加流入細胞之鈉離子的電流量，且並不改變鈉離子通道Nav1.4其活化(activation)與不活化(inactivation)特性。提高pipette solution中鈣離子濃度至10μM，鈣調素和突變型鈣調素均會增加鈉離子電流，而鈣調素會縮短鈉離子通道由不活化(inactivation)狀態再回到通道可再開啟的時間。若降低pipette solution中鈣離子濃度至0.2μM，其通道蛋白由不活化狀態恢復至通道可再開啓的時間則不受影響。此外，鈣調素在Nav1.1上亦有類似的調控情形，會增加鈉離子的電流。 經由西方點墨法(western blot)已及細胞免疫染色的分析，發現細胞膜上鈉離子通道的表現量有增加的現象。另外，在IQ motif上進行點突變亦會觀察到上述兩項改變。實驗結果顯示鈣調素可藉由感應胞內鈣離子濃度的變化進一步調控電壓依賴型鈉離子通道之特性。

Voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of action potentials in excitable cells. Calmodulin (CaM), a calcium sensor protein, regulates many types of ionic channels by binding to the highly conserved IQ motif at the intracellular C-terminal. Several reports have suggested that CaM has differential binding affinities with peptides containing the IQ motifs of various Na+ channels. However, it is not clear how CaM modulate the Na+ channel activities. In this report, we co-expressed CaM and Nav1.4 in 293T cells and measured the Na+ currents by patch-clamp technique in whole-cell mode. Both CaM and the Ca2+-binding deficient mutant, CaM1234, enhanced the current amplitude without changing the activation and inactivation properties. Elevating the Ca2+ concentration in the pipette solution to 10 μM, CaM and CaM1234 further increased the Na+ current; however, only CaM shortened the recovery time. While with 0.2 μM Ca2+ in the pipette solution, both CaM and CaM1234 enhanced the Na+ currents but had no effect on recovery time. Staining the expression level of Nav1.4 with a specific antibody, the amount of Nav1.4 at the plasma membrane was increaseed by CaM and CaM1234. Mutation in the IQ motif increased the Na+ current and recovery rates. CaM had a similar effect in enhancing the Nav1.1 currents. These findings suggest that CaM modulates VGSCs via the IQ motif in response to intracellular calcium concentration changes.