Pape P C, Jong D S, Chandler W K
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510-8026, USA.
J Gen Physiol. 1995 Aug;106(2):259-336. doi: 10.1085/jgp.106.2.259.
Sarcoplasmic reticulum (SR) Ca release was studied at 13-16 degrees C in cut fibers (sarcomere length, 3.4-3.9 microns) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the delta pH signal measured with phenol red by -beta/2. The value of beta, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 microM in the action-potential experiments and 2,544 microM in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of approximately 5%/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of approximately 3%/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7,000 and increase the value of spatially averaged myoplasmic free [Ca] by only 0.2 nM.
在13 - 16摄氏度下，对安装在双凡士林间隙室中的切断纤维（肌节长度为3.4 - 3.9微米）的肌浆网（SR）钙释放进行了研究。用含有20 mM乙二醇双四乙酸（EGTA）、1.76 mM钙和0.63 mM酚红的终池溶液平衡后，动作电位刺激的游离[Ca]瞬变的幅度和持续时间减小，这一操作似乎显著减少了肌钙蛋白结合的钙量。理论分析表明，在这些条件下，肌浆中游离[Ca]的增加预计局限于SR钙释放位点几百纳米范围内，且时间进程与释放基本匹配。此外，预计从SR释放的几乎所有钙都会迅速与EGTA结合，并以1:2的化学计量比与质子交换。因此，SR钙释放的时间进程可以通过将用酚红测量的ΔpH信号乘以 -β/2来估计。β值即肌浆的缓冲能力，是在与EGTA、酚红和fura - 2平衡的纤维中测定的；其平均值为22 mM/pH单位。SR的钙含量（以肌浆浓度表示）是通过一系列动作电位或耗尽电压阶跃释放出的钙总量来估计的；在动作电位实验中其平均值为2685微摩尔，在电压钳实验中为2544微摩尔。一个动作电位平均释放出SR钙含量的0.14，释放峰值速率约为5%/毫秒。在20毫秒后引发的第二个动作电位释放的钙量（以SR含量的分数表示）仅为第一个动作电位的0.6倍，这可能是因为第一个动作电位导致了钙释放的失活。在去极化电压阶跃到60 mV期间，钙释放速率迅速增加到约3%/毫秒的峰值，然后降至仅为峰值0.6倍的准稳态水平；这种下降也可能是由于钙释放的失活。通过小步去极化研究SR钙释放，这种去极化在7000个通道中打开的SR钙通道不超过一个，并且仅使空间平均的肌浆游离[Ca]值增加0.2 nM。
