Department of Pharmacology and Center for Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Neurosci. 2010 Mar 17;30(11):3904-11. doi: 10.1523/JNEUROSCI.4000-09.2010.
Changes in cellular cholesterol can affect exocytosis, but the influence of cholesterol in fusion pore kinetics is unclear. Using carbon fiber amperometry, we monitored quantal catecholamine release from rat chromaffin cells. To bypass any possible effect of cholesterol perturbation on ion channels or the colocalization of voltage-gated Ca(2+) channels with sites of exocytosis, exocytosis was stimulated via uniform elevation of cytosolic [Ca(2+)] (with whole-cell dialysis of a Ca(2+)-buffered solution). Under this condition, alterations of cellular cholesterol affected neither the mean number of amperometric events triggered per cell nor their quantal size and the kinetics of their main spike (which reflects the rapid release during and after rapid fusion pore dilation). In contrast, the reduction of cellular cholesterol shortened the "prespike foot" signals (which reflect the leakage of catecholamine via a semi-stable fusion pore) and reduced the proportion of "stand-alone foot" signals (which reflect the release via a flickering fusion pore that may close before it dilates significantly), whereas an oversupply of cholesterol had opposite effects. Acute extraction of cholesterol from the cytosol (via whole-cell dialysis of a cholesterol extractor) also shortened the prespike foot signals and reduced the proportion of stand-alone foot signals, but acute extracellular application of cholesterol extractor or "soluble" cholesterol had no effect. Our data raise the possibility that cholesterol molecules, particularly those in the cytoplasmic leaflet, helps to constrain the narrow waistline of a semi-stable fusion pore while it is flickering or before it starts to dilate rapidly.
细胞胆固醇的变化会影响胞吐作用，但胆固醇对融合孔动力学的影响尚不清楚。我们使用碳纤维安培法监测大鼠嗜铬细胞中儿茶酚胺的量子释放。为了避免胆固醇扰动对离子通道或电压门控 Ca(2+)通道与胞吐作用部位的共定位的任何可能影响，通过均匀升高细胞溶质 [Ca(2+)]（用 Ca(2+)缓冲溶液进行全细胞透析）来刺激胞吐作用。在这种条件下，细胞胆固醇的改变既不影响每个细胞引发的电化学测量事件的平均数量，也不影响它们的量子大小和主要尖峰的动力学（反映快速融合孔扩张期间和之后的快速释放）。相比之下，细胞胆固醇的减少缩短了“尖峰前足”信号（反映通过半稳定融合孔漏出儿茶酚胺），并降低了“独立足”信号的比例（反映通过可能在明显扩张之前关闭的闪烁融合孔释放），而胆固醇的过量供应则有相反的效果。细胞质中胆固醇的急性提取（通过全细胞透析胆固醇提取器）也缩短了尖峰前足信号，并降低了独立足信号的比例，但急性细胞外应用胆固醇提取器或“可溶性”胆固醇没有效果。我们的数据提出了这样一种可能性，即胆固醇分子，特别是细胞质小叶中的胆固醇分子，有助于限制半稳定融合孔在闪烁或开始快速扩张时的狭窄腰围。
