Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.

An increase in free Ca2+ triggers exocytosis in pituitary nerve terminals leading to an increase in membrane area and membrane capacitance. When Ca2+ is increased by step depolarization, an instantaneous capacitance increase during the first 80 ms is followed by a slow increase extending over several seconds. We measured capacitance changes associated with exocytosis and endocytosis in single pituitary nerve terminals internally perfused with high Ca2+. At 50 microM Ca2+ the capacitance increased by up to 2%/s, similar to the slow phase observed during depolarization. Our results indicate that at the site of fusion very high Ca2+ is required. Following exocytosis, large downward capacitance steps were measured, reflecting endocytosis of large vacuoles. These events were not abrupt but reflected a gradual decrease of fission pore conductance from 8 nS to < 40 pS during 500 ms, revealing the dynamics of individual fission pore closures. Above 300 pS, narrowing of the endocytotic fission pore was approximately 10 times slower than the previously reported expansion of the exocytotic fusion pore. The transition between 300 pS and 0 pS took approximately 200 ms, whereas it has been reported that the exocytotic fusion pore measured in mast cells opens from 0 to 280 pS in < 100 microseconds. The time course of closing of the fission pore may be explained by an exponential decrease in pore diameter occurring at a constant rate.

游离钙离子（free Ca²+）浓度升高可触发垂体神经末梢的胞吐作用（exocytosis），进而导致膜面积与膜电容（membrane capacitance）增加。当通过阶梯式去极化（step depolarization）提升钙离子浓度时，最初80毫秒内出现瞬时电容升高，随后伴随持续数秒的缓慢电容上升过程。我们对内部灌至高浓度钙离子的单个垂体神经末梢进行了检测，测量了与胞吐及内吞作用（endocytosis）相关的电容变化。在钙离子浓度为50 μM时，电容以最高2%每秒的速率升高，这与去极化过程中观察到的慢相变化一致。研究结果表明，在融合位点需要极高浓度的钙离子。胞吐作用发生后，我们检测到较大的电容阶跃式下降，这反映了大型囊泡的内吞过程。此类变化并非突然发生，而是表现为分裂孔（fission pore）电导在500毫秒内从8纳西门子（nS）逐渐降至40皮西门子（pS）以下，揭示了单个分裂孔闭合的动态过程。当分裂孔电导高于300 pS时，内吞分裂孔的收窄速率约为此前报道的胞吐融合孔（fusion pore）扩张速率的十分之一。分裂孔电导从300 pS降至0的过程耗时约200毫秒，而此前有研究显示，肥大细胞（mast cells）中的胞吐融合孔可在100微秒（microseconds）内从0扩张至280 pS。分裂孔的闭合时程可通过以恒定速率呈指数衰减的孔径变化来解释。