Simon B J, Klein M G, Schneider M F
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
J Gen Physiol. 1991 Mar;97(3):437-71. doi: 10.1085/jgp.97.3.437.
The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.
在含有两种钙指示剂（fura-2和抗吡唑啉酮III，即AP III）的电压钳制的青蛙骨骼肌纤维切段中，研究了肌浆网钙释放失活的稳态钙依赖性。fura-2荧光用于监测静息钙以及小去极化期间相对较小的钙瞬变。AP III吸光度信号用于监测大去极化期间较大的钙瞬变。从钙瞬变计算出肌浆网钙释放速率（Rrel）。使用不同幅度的200毫秒预脉冲将[Ca2+]提升至不同的稳态水平，以确定钙释放失活的平衡钙依赖性。每个预脉冲后跟随一个恒定的测试脉冲。测试脉冲期间峰值Rrel的抑制提供了预脉冲结束时释放失活程度的度量。失活的[Ca2+]依赖性表明，每个释放通道失活需要结合多个钙离子。在约0.3 microM的[Ca2+]时产生半最大失活。预脉冲持续时间和幅度的变化表明，峰值释放的抑制与钙释放的钙依赖性失活一致，而与钙耗竭不一致。使用不同幅度的测试脉冲确定失活程度时，获得了相同的失活钙依赖性。在随后的测试脉冲期间产生接近最大释放失活的预脉冲在测试脉冲期间未抑制膜内电荷移动，表明失活发生在钙释放电压传感器之后的步骤。为纤维固有的快速平衡钙结合位点假设的三组不同特性给出了略有不同的Rrel记录，但给出了非常相似但失活钙依赖性。因此，钙释放的平衡失活似乎是由[Ca2+]比静息水平适度增加并以陡峭的钙依赖性方式产生的。然而，即使在[Ca2+]明显升高期间，失活发展也相对缓慢，表明在初始钙结合步骤之后似乎发生了与钙无关的转变。
