Oberhauser A F, Monck J R, Fernandez J M
Department of Physiology and Biophysics, Mayo Clinic, Rochester, Minnesota 55905.
Biophys J. 1992 Mar;61(3):800-9. doi: 10.1016/S0006-3495(92)81884-2.
The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space. We can observe the formation of individual fusion pores and their subsequent dilation or closure by measuring the changes in the admittance of patch-clamped mast cells during GTP gamma S-stimulated exocytotic fusion. To investigate the molecular structure of the fusion pore, we have studied the temperature dependency of the rate constants for fusion pore formation and closure. An Arrhenius plot of the rate of fusion pore formation shows a simple linear relationship with an apparent activation energy of 23 kcal/mol. In contrast, the Arrhenius plot of the rate of closure of the fusion pore is discontinuous, with the break at approximately 13 degrees C. Above the break point, the rate of closure has a weak temperature dependence (7 kcal/mol), whereas below 13 degrees C the rate of closure is temperature independent. This type of temperature dependency is characteristic of events that depend on diffusion in a lipid phase that undergoes a fluid-solid phase transition. We propose that the formation of the fusion pore is regulated by the conformational change of a molecular structure with a high activation energy, whereas the closure of the fusion pore is regulated by lipids that become phase separated at 13 degrees C.
胞吐作用中最早发生的事件是形成融合孔，这是一种连接分泌颗粒内腔与细胞外空间的水性通道。我们可以通过测量GTPγS刺激的胞吐融合过程中膜片钳记录的肥大细胞导纳变化，来观察单个融合孔的形成及其随后的扩张或关闭。为了研究融合孔的分子结构，我们研究了融合孔形成和关闭速率常数的温度依赖性。融合孔形成速率的阿仑尼乌斯图显示出简单的线性关系，表观活化能为23千卡/摩尔。相比之下，融合孔关闭速率的阿仑尼乌斯图是不连续的，在大约13℃处有断点。在断点以上，关闭速率对温度的依赖性较弱（7千卡/摩尔），而在13℃以下，关闭速率与温度无关。这种温度依赖性是依赖于在经历液-固相变的脂质相中扩散的事件的特征。我们提出，融合孔的形成受具有高活化能的分子结构构象变化的调节，而融合孔的关闭受在13℃时发生相分离的脂质的调节。
