Calabrese Barbara, Tabarean Iustin V, Juranka Peter, Morris Catherine E
Department of Neurosciences, Ottawa Health Research Institute, Ottawa, Ontario K1Y 4E9, Canada.
Biophys J. 2002 Nov;83(5):2560-74. doi: 10.1016/S0006-3495(02)75267-3.
Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.
电压门控钙通道中的机械敏感性在健康细胞的钙信号传导中可能是一项优势，但在创伤期间可能成为一种负担。在HEK细胞中表达的重组N型通道显示出一系列机械反应。当在全细胞钳制下静水压使细胞膨胀时，电容不变，但峰值电流可逆地增加约1.5倍，这与细胞膨胀相关，而非施加的压力相关。此外，拉伸瞬时增加了开放态失活速率，不可逆地增加了关闭态失活速率，并使失活向左移位，而不影响激活曲线或速率。不可逆的机械反应被证明是电流衰减的机械加速成分；在细胞贴附记录中它们不明显，然而，可逆的拉伸诱导的峰值电流增加持续存在。单独表达或与N型通道（α(1B)和两个辅助亚基）共表达并与增强N型电流的拉伸共同刺激时，T型通道（仅α(1I)亚基）对机械不敏感。连同细胞贴附结果，这种差异效应表明N型机械敏感性不依赖于记录情况。T型电流对拉伸不敏感表明N型机械反应可能源于主要/辅助亚基相互作用。然而，在单通道记录中，无论α(1B)亚基单独表达还是与辅助亚基一起表达，N型电流都表现出可逆的拉伸诱导的NP(o)增加。这些发现为钙电流机械敏感性的分子剖析奠定了基础。
