Evidence for Ca2+ activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex
Evidence for Ca2+ activation and inactivation sites on the luminal side of the cardiac ryanodine receptor complex
复制标题
DOI：
10.1161/01.res.87.3.201
复制
发表时间：
2000-08-04
影响因子：
20.1
通讯作者：
Sitsapesan, R
中科院分区：
文献类型：
--
作者：
Ching, LL;Williams, AJ;Sitsapesan, R
我们使用胰蛋白酶消化来确定 Ca2+ 是否可以调节肌浆网 (SR) 内腔内的心脏兰尼碱受体 (RyR) 通道门控，或者 Ca2+ 是否必须首先流过该通道并通过位于胞质的结合位点发挥作用。将心脏 RyR 纳入双层，并将胰蛋白酶应用于双层的管腔侧。我们发现，在暴露于管腔胰蛋白酶之前，通过将管腔[Ca2+]从10μmol/L提高到1mmol/L，RyR的开放概率增加，而在暴露于管腔胰蛋白酶之后，增加管腔[Ca2+]降低了开放概率。使用热灭活的胰蛋白酶未观察到 RyRs 对管腔 Ca2+ 响应的改变，表明 RyR 通道复合物上管腔位点的消化是造成这种变化的原因。我们的结果为管腔 Ca2+ 激活和抑制位点的存在提供了强有力的证据，并表明胰蛋白酶消化导致对管腔 Ca2+ 激活位点的选择性损伤，而不影响管腔 Ca2+ 失活位点。我们认为，腔内 [Ca2+] 的变化将能够从 SR 腔内调节 RyR 通道门控，因此除了胞质 Ca2+ 之外，还对 RyR 通道门控提供第二种 Ca2+ 调节作用。这种腔内 Ca2+ 调节机制可能是增强 SR Ca2+ 释放的重要因素，在心肌细胞中观察到 SR Ca2+ 释放增强，以响应内部 SR [Ca2+] 的增加。
We have used tryptic digestion to determine whether Ca2+ can regulate cardiac ryanodine receptor (RyR) channel gating from within the lumen of the sarcoplasmic reticulum (SR) or whether Ca2+ must first flow through the channel and act via cytosolically located binding sites. Cardiac RyRs were incorporated into bilayers, and trypsin was applied to the luminal side of the bilayer. We found that before exposure to luminal trypsin, the open probability of RyR was increased by raising the luminal [Ca2+] from 10 mu mol/L to 1 mmol/L, whereas after luminal trypsin exposure, increasing the luminal [Ca2+] reduced the open probability. The modification in the response of RyRs to luminal Ca2+ was not observed with heat-inactivated trypsin, indicating that digestion of luminal sites on the RyR channel complex was responsible, Our results provide strong evidence for the presence of luminally located Ca2+ activation and inhibition sites and indicate that trypsin digestion leads to selective damage to luminal Ca2+ activation sites without affecting luminal Ca2+ inactivation sites. We suggest that changes in luminal [Ca2+] will be able to regulate RyR channel gating from within the SR lumen, therefore providing a second Ca2+-regulatory effect on RyR channel gating in addition to that of cytosolic Ca2+. This luminal Ca2+-regulatory mechanism is likely to be an important contributing factor in the potentiation of SR Ca2+ release that is observed in cardiac cells in response to increases in intra-SR [Ca2+].