大鼠酸感受离子通道亚基2a表达质粒的构建及生物学特性考察

Construction of expression plasmid rat acid sensing ion channel subunit 2a and its biological characteristics research

目的构建大鼠酸感受离子通道亚基2a(acid-sensingion channel subunit 2a,ASIC2a)的表达质粒并研究其组成的同聚体离子通道的生物学特性。方法使用分子生物学技术构建大鼠ASIC2a亚基表达质粒;通过体外转录技术,使编码ASIC2a亚基的cRNA在爪蟾卵母细胞内表达并在膜表面形成同聚体离子通道;使用双电极电压钳技术研究ASIC2a的生物学特性。结果在注射ASIC2a亚基cRNA的爪蟾卵母细胞上,降低胞外液pH值可诱导出内向电流。H+诱发的ASIC2a内向电流具有稳态失活成分可被氨氯吡咪可逆性阻断,其pH50为5.12。提高胞外Ca2+浓度可降低H+诱发的电流幅度,其IC50为11.98 mmol.L-1。当细胞外液中无Na+时,H+基本上不能诱发出内向电流;当同时去除细胞外液中Na+和K+时,H+可诱发出外向电流。结论成功构建ASIC2a表达质粒;ASIC2a除了对Na+通透外,对K+也有一定的通透性,胞外Ca2+抑制ASIC2a孔道的开放。

Objective To construct the expression plasmid of rat acid sensing ion channel subunit 2a （ASIC2a） and study the biological characteristics of its homopolymer ion channels. Methods The expression plasmid of rat ASIC2a was constructed by using molecular biology technique. The cRNA which encodes ASIC2a was injected into Xenopus laevis oocyte with two-electrode voltage clamp technique, The cRNA of ASIC2a expressed and formed homopolymers in Xenopus laevis oocyte membrane, through that we could study the biological characteristics of rat ASIC2a. Results In Xenopus laevis oocytes injected with cRNA of ASIC2a, the inward currents could be induced when extracellular pH was decreased. The inward currents of ASIC2a had steady-state inactivation and could be blocked reversibly by amiloride with pHs0 value of 5.12. Ca^2 ＋ had inhibitive effects on the currents of ASIC2a with IC50 value of 11.98 mmol· L^- 1. There was no inward currents when the extracellular fluids without Na^＋ and H^＋-induced currents changed to outward when the extracellular fluids without both of Na^＋ and K^＋ . Conclusions The expression plasmid of ASIC2a is constructed successfully. Beside the permeability to Na^＋, ASIC2a also has the permeability to K^＋. The amplitude of currents can be decreased by extracellular Ca^2＋.