R371H和P266T位点突变对ATP敏感性钾通道特性的影响

The effects of site-directed mutation on characters of ATP sensitive potassium channel

目的研究R371H和P266T两个位点突变对ATP敏感性钾通道特性的影响,揭示上述位点突变导致心律失常的机制。方法用人胚肾细胞表达Kiv6．2野生型、R371H和P266T位点突变后的通道,用膜片钳技术研究突变后胞内pH对ATP敏感性变构调节的变化。结果野生型、P266T、R371H均成功记录到内向整流性K+电流。暴露于不同的pH中时,野生型通道的半数电流抑制ATP浓度(IC50)在pH 6．8时较pH 7．4时显著增大(70 vs 22μmol／L,P<0．05),ATP浓度-电流曲线显著右移; R371H和P266T的IC50在pH 6．8与pH 7．4时差异无统计学意义。结论胞内pH对ATP敏感性变构调节的丧失,可能是R371H和P266T位点突变时导致心律失常的重要机制。

Objectives To study the effects of site-directed mutation R371H and P266T on characters of ATP sensitive potassium channel （ KATP ） in order to reveal the arrhythmogenic mechanism of the two sites mutation. Methods Wild-type （wt）, R371H or P266T mutant of Kit6.2 cDNA plus SUR2A were expressed in human embryonic kidney cells. Standard patch-clamp technique was used to study the allosteric modulation of ATP sensitive by intracellular pH. Results Inward rectifier potassium currents were recorded in wt, R371H, P266T successfully. While exposed in different pH, half current inhibition of ATP concentration （ IC5o ） of wt in pH6. 8 was significant higher than that in pH7.4 （70 vs 22μmol/L, P 〈 0.05 ） , and the ATP-current curve was shifted to higher ATP concentration. The IC50 of both R371H and P266T in pH 6. 8 and pH 7.4 had no significant difference. Conclusion The loss of allosteric modulation of ATP sensitive by intracellular pH maybe an important arrhythmogenic mechanism of R371H and P266T mutation.