Departamento de Biofísica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay.
J Muscle Res Cell Motil. 2010 Aug;31(2):81-92. doi: 10.1007/s10974-010-9212-y. Epub 2010 Jun 11.
Two procedures to inhibit Ca(2+) release designed to differentiate between local and common pool mechanisms for the Ca(2+) dependent, fast inactivation of Ca(2+) release in skeletal muscle of the frog were compared. Inhibition by voltage dependent inactivation of Ca(2+) release, without modification of the single channel current of the Ryanodine Receptor (RyR) and the [Ca(2+)] close to the open pore, produced a reduction in the rate of inactivation linearly related to the reduction in the peak of Ca(2+) release flux. Linear fits in the individual fibers were performed, giving average values (+/-SEM, N = 8) of the best fit parameters of 5.75 x 10(-3) +/- 7.35 x 10(-4 )microM(-1) for the slope and 0.07 +/- 0.015 ms(-1) for the ordinate intercept. Inhibition of Ca(2+) release by reducing the Ca content of the sarcoplasmic reticulum (SR) involves reduction of the Ca(2+) current through the single RyR. The reduction in rate of inactivation also followed linearly the reduction in Ca(2+) peak release flux. The average values (+/-SEM) of the best fit parameters of linear fits were 14 x 10(-3) +/- 3.76 x 10(-3 )microM(-1) and 0.019 +/- 0.006 ms(-1) (N = 7) for slope and ordinate intercept respectively. The differences between both parameters were statistically significant (by t test, at P = 0.05). The extent of inactivation, measured by the peak/final Ca(2+) release flux ratio, was differentially affected by the two procedures. Inhibition by voltage dependent inactivation, despite slowing down the fast inactivation, increased the peak/final Ca(2+) release flux ratio. In contrast, depletion of the SR reticulum reduced it. If the fast inactivation is driven by the high [Ca(2+)] attained locally, close to the open pore of the RyR, the inhibition of Ca(2+) release due to voltage dependent inactivation should not modify the rate of inactivation while inhibition by SR Ca(2+) depletion should reduce it. A process driven by [Ca(2+)] in a common pool should depend on the overall Ca(2+) release independently of how it was modified. In this case both inhibitory procedures should reduce the inactivation rate similarly. Our findings are generally consistent with a common pool process. The differences between the two protocols could be understood if the organization of RyR in junctional and parajunctional release units is considered.
两种抑制 Ca(2+) 释放的方法被用来区分 Ca(2+) 依赖性、快速失活 Ca(2+) 释放的局部和共同池机制，该机制存在于青蛙的骨骼肌中。电压依赖性失活对 Ca(2+) 释放的抑制作用，不会改变 RyR 的单通道电流和靠近开放孔的 [Ca(2+)]，导致失活速率的线性降低与 Ca(2+) 释放通量峰值的降低成正比。对单个纤维进行线性拟合，得出最佳拟合参数的平均值（+/-SEM，N = 8）为斜率 5.75 x 10(-3) +/- 7.35 x 10(-4) microM(-1)，纵坐标截距 0.07 +/- 0.015 ms(-1)。通过降低肌浆网（SR）中的 Ca 含量来抑制 Ca(2+) 释放，涉及到通过单个 RyR 的 Ca(2+) 电流的减少。失活速率的降低也与 Ca(2+) 峰值释放通量的降低呈线性关系。线性拟合的最佳拟合参数的平均值（+/-SEM）分别为斜率 14 x 10(-3) +/- 3.76 x 10(-3) microM(-1)和纵坐标截距 0.019 +/- 0.006 ms(-1)（N = 7）。两个参数之间的差异具有统计学意义（通过 t 检验，P = 0.05）。通过峰值/最终 Ca(2+) 释放通量比值测量的失活程度，受到两种方法的不同影响。尽管电压依赖性失活减缓了快速失活，但它增加了峰值/最终 Ca(2+) 释放通量的比值。相比之下，SR 网状物的耗竭降低了它。如果快速失活是由靠近 RyR 开放孔的局部高 [Ca(2+)] 驱动的，那么由于电压依赖性失活而导致的 Ca(2+) 释放抑制不应改变失活速率，而由于 SR Ca(2+) 耗竭导致的失活速率应该降低。由共同池中的 [Ca(2+)] 驱动的过程应该取决于整体 Ca(2+) 释放，而与如何改变无关。在这种情况下，两种抑制程序都应该以相似的方式降低失活速率。我们的发现与共同池过程基本一致。如果考虑 RyR 在连接和副连接释放单元中的组织方式，那么两种方案之间的差异是可以理解的。
