Yue Peng, Zhang Yong, Du Zhimin, Xiao Jing, Pan Zhenwei, Wang Ning, Yu Haiyan, Ma Wencai, Qin Hong, Wang Wen-Hui, Lin Dao-Hong, Yang Baofeng
Department of Pharmacology, Harbin Medical University, Bio-Pharmaceutical Key Laboratory of Heilongjiang Province, Harbin, China.
Cell Physiol Biochem. 2006;17(3-4):129-36. doi: 10.1159/000092074. Epub 2006 Mar 14.
We used Western blot analysis to examine the expression of connexin 43 and M2/M3 acetylcholine muscarinic receptors (mAChR) and their interaction in ventricular myocytes from control and the ischemic heart. We confirmed that the connexin 43 and M2/ M3-mAChR were expressed in ventricular myocytes. Moreover, we showed that M3-mAChR was expressed in non-glycosylated (72 kDa) and glycosylated forms (115 kDa). Immunostaining showed that connexin 43 is closely associated with M3-mAChR in parts of cell membranes of myocytes. Immunoprecipitation of lysate of cardiac myocytes with M2/M3-mAChR antibody pulled down a 44 kDa protein recognized by connexin 43 antibody. Ischemia increased the expression of M3-mAChR in myocytes. The ischemiainduced increase in the M3-mAChR expression was specific because ischemia did not affect the expression of M1, M2, M4 and M5- mAChR in the heart. On the other hand, ischemia decreased the expression of connexin 43 in myocardium. We also examined the effect of ischemia on the interaction between M2/M3-mAChR and connexin 43. Ischemia suppressed the association of M3-mAChR with connexin 43 but did not affect the association of connexin 43 with M2-mAChR. Administration of choline before ischemia not only partially restored the expression of connexin 43 but also attenuated the ischemia-induced suppression of the association between connexin 43 and M3-mAChR. We conclude that connexin 43 interacts with M2/M3-mAChR and that ischemia specifically impairs the association between M3-mAChR and connexin 43.
我们采用蛋白质免疫印迹分析来检测对照组和缺血心脏心室肌细胞中连接蛋白43及M2/M3型毒蕈碱型乙酰胆碱受体（mAChR）的表达及其相互作用。我们证实连接蛋白43及M2/M3-mAChR在心室肌细胞中表达。此外，我们发现M3-mAChR以非糖基化形式（72 kDa）和糖基化形式（115 kDa）表达。免疫染色显示，连接蛋白43在部分心肌细胞膜中与M3-mAChR紧密相关。用M2/M3-mAChR抗体对心肌细胞裂解物进行免疫沉淀，沉淀出一种可被连接蛋白43抗体识别的44 kDa蛋白。缺血增加了心肌细胞中M3-mAChR的表达。缺血诱导的M3-mAChR表达增加具有特异性，因为缺血不影响心脏中M1、M2、M4和M5-mAChR的表达。另一方面，缺血降低了心肌中连接蛋白43的表达。我们还研究了缺血对M2/M3-mAChR与连接蛋白43之间相互作用的影响。缺血抑制了M3-mAChR与连接蛋白43的结合，但不影响连接蛋白43与M2-mAChR的结合。缺血前给予胆碱不仅部分恢复了连接蛋白43的表达，还减弱了缺血诱导的连接蛋白43与M3-mAChR结合的抑制作用。我们得出结论，连接蛋白43与M2/M3-mAChR相互作用，且缺血特异性损害M3-mAChR与连接蛋白43之间的结合。
