Tang Wang-Xian, Chen Ya-Fei, Zou Ai-Ping, Campbell William B, Li Pin-Lan
Research Institute of Liver Disease, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Am J Physiol Heart Circ Physiol. 2002 Apr;282(4):H1304-10. doi: 10.1152/ajpheart.00843.2001.
cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.
环ADP核糖（cADPR）作为第二信使激活血管平滑肌细胞肌浆网（SR）的兰尼碱受体（RyRs）并动员细胞内钙离子（Ca2+）。然而，介导cADPR作用的机制尚不清楚。本研究旨在确定FK-506结合蛋白12.6（FKBP12.6），一种RyRs的辅助蛋白，是否在cADPR诱导的RyRs激活中发挥作用。用抗FKBP12抗体免疫印迹法在牛冠状动脉平滑肌（BCASM）和培养的CASM细胞中检测到一种12.6 kDa的蛋白，该抗体也与FKBP12.6发生反应。利用平面脂质双层钳制技术，FK-506（0.01 - 10 μM）显著增加了重组自CASM肌浆网的RyR/Ca2+释放通道的开放概率（NP(O)）。抗FKBP12抗体预处理可消除FK-506诱导的RyR/Ca2+释放通道激活。RyRs激活剂cADPR（0.1 - 10 μM）显著增加了RyR/Ca2+释放通道的活性。在FK-506存在的情况下，cADPR并未进一步增加RyR/Ca2+释放通道的NP(O)。添加抗FKBP12抗体也完全阻断了cADPR诱导的这些通道激活，用FK-506预孵育并随后梯度离心去除FKBP12.6消除了cADPR诱导的RyR/Ca2+释放通道NP(O)增加。我们得出结论，FKBP12.6在介导cADPR诱导的BCASM肌浆网RyR/Ca2+释放通道激活中起关键作用。
