低钾条件下TWIK-1双孔钾通道对小鼠心肌细胞膜电位的影响及其机制研究

Effect of TWIK-1 two-pore potassium channel on membrane potential of mouse cardiac myocytes at sub-physiological extracellular potassium con⁃centration

目的:探讨在细胞外低钾条件下TWIK-1双孔钾通道对小鼠HL-1心肌细胞膜电位的影响及其分子机制。方法:构建载有人TWIK-1或TWIK-1-T118i突变体基因序列的慢病毒载体。通过病毒转染的方法使中国仓鼠卵巢细胞(CHO细胞)或HL-1小鼠心肌细胞表达TWIK-1或TWIK-1-T118i突变体。通过膜片钳记录在细胞外正常钾(5.4 mmol/L)及低钾(2.7 mmol/L)条件下的膜电位及全细胞电流。结果:电流钳结果显示,在2.7 mmol/L钾条件下,表达人TWIK-1通道的CHO细胞及HL-1小鼠心肌细胞膜电位呈现去极化,当细胞外液的钠离子被替换为N-甲基-D-葡萄糖胺(NMDG)时,膜电位去极化现象消失;而表达人TWIK-1-T118i突变体通道的CHO细胞及HL-1小鼠心肌细胞膜电位呈现超极化。电压钳结果显示,在2.7 mmol/L钾条件下,在表达人TWIK-1通道的CHO细胞及HL-1小鼠心肌细胞可记录到内向阳离子电流,当细胞外液的钠离子替换为NMDG时,该内向阳离子电流消失。结论:在细胞外低钾条件下,TWIK-1通道介导的内向钠离子电流能够引起HL-1小鼠心肌细胞膜电位发生去极化。

AIM:To investigate the effect of TWIK-1 two-pore potassium channel on membrane potential of HL-1 mouse cardiac myocytes at sub-physiological extracellular potassium concentration([K^(+)]_(e)).METHODS:Chinese hamster ovary cells(CHO cells)or HL-1 mouse cardiac myocytes were transfected with lentiviral particles carrying human TWIK-1 or TWIK-1-T118i gene.The membrane potentials or whole-cell currents of the cells in each group were recorded by patch clamp at 5.4 mmol/L[K^(+)]_(e)and 2.7 mmol/L[K^(+)]_(e).RESULTS:Current clamp results showed that the mem-brane potentials of CHO cells or HL-1 mouse cardiac myocytes with ectopic expression of human TWIK-1 channel were de-polarized at 2.7 mmol/L[K^(+)]_(e),and the phenomenon was eliminated when extracellular Na+was replaced with N-methyl-D-glucamine(NMDG),while the membrane potentials of CHO cells or HL-1 mouse cardiac myocytes with ectopic expres-sion of human TWIK-1-T118i mutant channel were hyperpolarized at 2.7 mmol/L[K^(+)]_(e).Voltage clamp results showed that CHO cells or HL-1 mouse cardiac myocytes with ectopic expression of human TWIK-1 channel displayed whole-cell in-ward cation currents at 2.7 mmol/L[K^(+)]_(e),and the inward currents was abolished when extracellular Na+was replaced with NMDG.CONCLUSION:The inward Na+currents conducted by TWIK-1 channel induce membrane potential depolariza-tion of HL-1 mouse cardiac myocytes at sub-physiological[K^(+)]_(e).