布比卡因对KG1a细胞的中电导钙激活的钾通道的调控作用

Bupivacaine Inhibits the Activity of Intermediate Conductance Ca2+-Activated K+ Channels in KG1a Cells

目的:探讨局部麻醉药布比卡因(Bupivacaine)对中电导钙激活的钾通道(IKCa)的调控作用。方法:体外培养KG1a细胞,PCR检测IKCa基因KCNN4和小电导钙激活的钾通道基因KCNN1-3的表达,全细胞膜片钳方法检测游离Ca2+浓度为1mmol/L的电极内液条件下,破膜前后钾电流变化,并分别观察bupivacaine和IKCa阻断剂Clotrimazole对其的影响。结果:KG1a细胞表达KCNN4mRNA,但不表达KCNN1-3基因;在全细胞记录模式下,刚破膜时记录到微弱钾电流,3min后电流幅度明显增大,同时细胞膜电位由(-22.4±2.3)mV超极化至(-64.3±3.7)mV(P<0.01);IKCa阻断剂Clotrimazole(1mmol/L)作用后,最大电流下降至对照的(34±5)%(P<0.05);bupivacaine(1mmol/L)作用后的电流下降至对照的(28±5)%(P<0.05),该抑制作用可以部分被洗脱。挥发性麻醉剂halothane作用后的KCa电流下降至对照的(30±4)%(P<0.05),但BKCa通道抑制剂四乙铵(TEA,1mmol/L)对KCa无明显抑制作用(P>0.05)。结论:IKCa电流是KG1a细胞的主要KCa电流成分,bupivacaine可以显著抑制IKCa电流。

Objective:To investigate the effect of local anesthetic bupivacaine on the activity of intermediate conductance Ca2+-activated K+channels(IKCa)of KG1 acells.Methods:mRNA expression of IKCa gene KCNN4,and small conductance K+channels(SKCa)gene KCNN1-3 on KG1 acells was determined with PCR,and whole-cell patch-clamp technique was used to record KCa current elicited by 1 mmol/L free Ca2+-containing pipette solution in KG1 acells.Results:PCR experiment showed that KG1 acells expressed only mRNA of KCNN4,but not KCNN1-3.Immediately after the rupture of cell membrane,we recorded a small current,and after diluted with 1 mmol/L free Ca2+-containing pipette solution for 3 min,a robust current was induced,with a reversal potential about-70 mV.This current could be inhibited by 1 mmol/L Clotrimazole,an IKCa blocker,but not by BKCa blocker TEA.The membrane potential of KG1 acells was also hyperpolarized from(-22.4±2.3)mV to(-64.3 ± 3.7)mV(P<0.01)after dilution with1 mmol/L free Ca2+-containing pipette solution.Bupivacaine(1 mmol/L)inhibited this current to(28±5)% of control(P<0.05).In addition,this current was also significantly inhibited by volatile anesthetic halothane at 1 mmol/L.Conclusion:IKCa is the major KCa channel in KG1 a cells,and bupivacaine can potently inhibit IKCa channels on KG1 acells.