Marengo Fernando D
Department of Physiology, UCLA, School of Medicine, Los Angeles, CA 90095, USA.
Cell Calcium. 2005 Aug;38(2):87-99. doi: 10.1016/j.ceca.2005.06.006.
The relationship between the localized Ca(2+) concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca(2+) imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca(2+) gradients and small "synchronous" increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca(2+) concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and K(d) = 1.4 microM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca(2+) gradients collapsed. Raising the pipette Ca(2+) concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca(2+)-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 microM and vesicles are mobilized to this pool in a Ca(2+)-dependent manner.
利用脉冲激光钙成像和膜电容测量技术，在膜片钳记录的肾上腺嗜铬细胞中研究了局部钙浓度与去极化诱导的胞吐作用之间的关系。短暂的去极化电压阶跃在脉冲期间诱导了钙梯度并使电容出现小幅度的“同步”增加。较长的脉冲增加了电容变化，在16飞法时达到饱和，这表明存在一小部分随时可释放的、准备好融合的囊泡池。以膜下薄（100纳米）壳层中估计的钙浓度为横坐标、电容变化为纵坐标绘制的希尔图得出n = 2.3和K(d) = 1.4微摩尔。重复刺激引发了更复杂的胞吐模式：早期脉冲诱导同步电容增加，但在五个或更多脉冲后，同步反应出现易化，并且随着陡峭的膜下钙梯度消失，脉冲之间的电容持续逐渐增加（异步胞吐）。提高移液管中的钙浓度导致同步反应的早期易化和异步胞吐的早期出现。我们利用这些数据建立了一个去极化诱导胞吐作用的动力学模型，其中囊泡的钙依赖性融合发生在一个亲和力为1 - 2微摩尔的小的即时可释放池中，并且囊泡以钙依赖性方式被动员到这个池中。
