膜蛋白LRRC8A在小鼠心肌细胞的表达及电生理特性

Expression and electrophysiological properties of leucine rich repeat containing 8 family number A in mouse cardiomyocytes

目的探讨容积敏感性氯通道蛋白主要成分LRRC8A在小鼠心肌细胞的表达及电生理特性。方法采用原代培养的小鼠乳鼠心肌细胞,上海吉玛公司合成4个不同LRRC8A的小干扰RNA(siRNA),片段依次为A组、B组、C组和D组,另将1个不包含siRNA的片段作为NC组。将含LRRC8AsiRNA片段的腺病毒感染心肌细胞,用等渗、低渗、低渗加DCPIB、低渗加siRNA外液灌流。RT-PCR法和Western Blot法检测各组LRRC8A在心肌细胞的表达;应用膜片钳检测各种状态下LRRC8A电生理特性(I_(Cl,vol))的变化。结果心肌细胞能正常表达LRRC8A,腺病毒成功感染细胞后,与NC组比较,A、B、C和D组LRRC8A mRNA及蛋白表达量均降低(P<0.05)。与等渗外液比较,低渗外液I_(Cl,vol)明显升高,与低渗外液比较,低渗加DCPIB外液的I_(Cl,vol)降低,抑制率达到(96.48±2.63)%(P<0.01),与低渗外液比较,低渗加siRNA外液的电流密度值下降达(83.04±8.41)%(P<0.01)。结论 LRRC8A是小鼠心肌细胞膜重要的组成成分,是容积敏感性氯通道的重要构成蛋白。

Objective To study the expression and electrophysiological properties of LRRC8 A,a main component of the volume-sensitive chloride channel,in mouse cardiomyocytes.MethodsPrimarily cultured mouse cardiomyocytes were divided into groups A-D and control group according to whether they contain LRRC8 AsiRNA fragments or not.The expression of LRRC8 Ain mouse cardiomyocytes was detected by RT-PCR and Western blot respectively.The electrophysiological properties of LRRC8 Ain mouse cardiomyocytes were assayed by patch clamping.Results The LRRC8 Awas normally expressed in mouse cardiomyocytes.The expression level of LRRC8 A mRNA was significantly lower in groups A-D than in control group after mouse cardiomyocytes were infected with adenoviruses（P〈0.05）.The volume of electrophysiological properties of LRRC8 A was significantly larger in mouse cardiomyocytes lavaged with hypotonic solution than in those lavaged with isotonic solution and was significantly smaller in mouse cardiomyocytes lavaged with hypotonic solution＋DCPIB than in those lavaged with hypotonic solution with an inhibition rate of 96.48% ±2.63%.The rate of current density decreased by 83.04% ±8.41% in mouse cardiomyocytes lavaged with hypotonic solution＋siRNA compared with that in those lavaged with hypotonic solution（P〈0.01）.Conclusion LRRC8 Ais an important component of volume-sensitive chloride channel in membranes of mouse cardiomyocytes.