部分纯化的牛心细胞质提取物对心肌细胞膜“run-down”L-型钙通道活性的恢复作用

Effects of Partial Purified Cytoplasmic Extract of Bovine Heart to Recover L-type Ca^(2+) Channel Activity after "Run-down"

目的研究牛心细胞质部分纯化提取物及钙调蛋白激酶Ⅱ(CaMKⅡ)对大鼠心肌细胞膜L-型钙通道活性的调节作用。方法采用常规的蛋白提取方法制备牛心细胞质提取物,离子交换方法进行部分纯化。应用膜片钳制技术的细胞贴附和膜内向外记录模式记录大鼠心室肌细胞单通道钙电流,观察牛心细胞质部分纯化提取物和CaMKⅡ对"run-down"L-型钙通道活性的恢复作用。Western blot方法验证活性蛋白。结果膜内向外记录模式下,L-型钙通道出现"run-down"现象,活性下降至细胞贴附记录模式的(0.46±0.15)%;牛心细胞质部分纯化提取物使"run-down"的通道活性恢复至细胞贴附记录模式的(44.29±13.51)%(P<0.01),加入CaMKⅡ抑制剂KN-62(1μmol/L)后活性仅恢复至(1.30±0.94)%,与未加抑制剂组相比差异有统计学意义(P<0.05);Western blot方法验证牛心细胞质部分纯化提取物中含有CaMKⅡ;基因重组融合蛋白法制备的CaMKⅡ也可将"run-down"后的钙通道活性恢复至细胞贴附记录模式的(71.59±14.76)%(P<0.05)。结论牛心细胞质部分纯化提取物对"run-down"钙通道活性具有恢复作用,该作用可能与CaMKⅡ密切相关。

Objective To investigate the effects of partial purified cytoplasmic extract and calmodulin-dependent protein kinase Ⅱ （CaMKI- I） in the regulation of the activities of L-type Ca2＋ channel in myocardial cell membrane of rats. Methods Cytoplasmic extract was pre- pared fuml bovine heart with routine method, and purified partially with ion-exchange. Cell attached and inside out mode of patch-clamp teehnique was employed to record the single Ca2＋ current in the rat ventricular myocytes, and observe the effects of partial purified cytoplasmic extract and CaMK Ⅱ on recovering the activity of L-type Ca2＋ channel. Western blot was used to verify the target active protein in the extract. Results Under the inside out mode,the L-type Ca2＋ channel showed a ＂run-down＂ phenomenon, and its activity decreased to （0.46± 0.15 ）% recorded under the cell-attached mode. The partial purified cytoplasmic extract recovered L-type Ca2＋ channel activity to （44.29± 13.51 ）% under the cell-attached mode （P 〈 0.01 ）. After KN-62 （ 1 μmol/L）,a CaMK Ⅱ inhibitor,was added,the L-type Ca2＋ channel ac- tivity reduced to （ 1.30±0.94）%,which was significant different comparing with non-KN-62 adding group（P 〈 0.05）. CaMK II was detected in partial purified cytoplasmic extract with western blot method. CaMK Ⅱ prepared with gene-recombination fusion-protein method could also reeover ＂run-down＂ L-type Ca2＋ channel aetivity to （71.59＋14.76）% under the cell-attached mode （P 〈 0.05 ）. Conclusion The partial purified cytoplasmic extract could recover L-type Ca2＋ ehannel activity after ＂run-down＂ whose effect might be related to CaMK II activity.