Györke Inna, Hester Nichole, Jones Larry R, Györke Sandor
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430-6551, USA.
Biophys J. 2004 Apr;86(4):2121-8. doi: 10.1016/S0006-3495(04)74271-X.
The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.
肌浆网（SR）内的钙离子水平是心肌中钙离子释放通道/雷诺丁受体（RyR）功能活性的重要决定因素。然而，管腔钙对RyR调节的分子基础仍 largely 未知。在本研究中，我们研究了心肌 SR 管腔辅助蛋白肌集钙蛋白（CSQ）、三联蛋白 1 和连接蛋白在形成心肌 RyR 的管腔钙传感器中的潜在作用。使用 Cs⁺作为电荷载体，在 MgATP 存在的情况下，对掺入脂质双层的单个 RyR 通道进行记录，这些通道来自 SR 囊泡或纯化的 RyR 制剂。将管腔[Ca]从 20 μM 提高到 5 mM 可增加 SR 囊泡中天然 RyR 的开放通道概率（Po），但对纯化的 RyR 则无此作用。向纯化通道的管腔侧添加 CSQ 对 Po 没有显著影响，也未恢复 RyR 对管腔钙的反应能力。当向纯化通道的管腔侧添加三联蛋白 1 和连接蛋白时，RyR 的 Po 显著增加；然而，通道对管腔[Ca]的变化仍无反应。在与三联蛋白 1 和连接蛋白重新结合的 RyR 中，添加管腔 CSQ 会导致活性显著降低。与所有三种蛋白质重新结合后，RyR 通过增加其 Po 对管腔[Ca]的升高做出反应。这些结果表明，CSQ、三联蛋白 1 和连接蛋白的复合物赋予 RyR 管腔钙敏感性。CSQ 显然作为管腔钙传感器，在低管腔[Ca]时抑制通道，而三联蛋白 1 和/或连接蛋白可能是介导 CSQ 与 RyR 相互作用所必需的。
