Department of Chemistry, University of Washington, Seattle, Washington, USA.
Biophys J. 2011 Oct 5;101(7):1580-9. doi: 10.1016/j.bpj.2011.08.032.
Uptake of neurotransmitters into synaptic vesicles is driven by the proton gradient established across the vesicle membrane. The acidification of synaptic vesicles, therefore, is a crucial component of vesicle function. Here we present measurements of acidification rate constants from isolated, single synaptic vesicles. Vesicles were purified from mice expressing a fusion protein termed SynaptopHluorin created by the fusion of VAMP/synaptobrevin to the pH-sensitive super-ecliptic green fluorescent protein. We calibrated SynaptopHluorin fluorescence to determine the relationship between fluorescence intensity and internal vesicle pH, and used these values to measure the rate constant of vesicle acidification. We also measured the effects of ATP, glutamate, and chloride on acidification. We report acidification time constants of 500 ms to 1 s. The rate of acidification increased with increasing extravesicular concentrations of ATP and glutamate. These data provide an upper and a lower bound for vesicle acidification and indicate that vesicle readiness can be regulated by changes in energy and transmitter availability.
神经递质被摄取到突触小泡中是由跨小泡膜建立的质子梯度驱动的。因此，突触小泡的酸化是小泡功能的关键组成部分。在这里，我们介绍了从分离的单个突触小泡中测量的酸化率常数。从小鼠中纯化出的小泡表达了一种融合蛋白，称为 SynaptopHluorin，它是由 VAMP/synaptobrevin 与 pH 敏感的超亮绿色荧光蛋白融合而成。我们对 SynaptopHluorin 荧光进行了校准，以确定荧光强度与内部小泡 pH 值之间的关系，并使用这些值来测量小泡酸化的速率常数。我们还测量了 ATP、谷氨酸和氯离子对酸化的影响。我们报告的酸化时间常数为 500ms 到 1s。酸化的速率随着细胞外 ATP 和谷氨酸浓度的增加而增加。这些数据为小泡酸化提供了一个上限和下限，并表明小泡的准备状态可以通过能量和递质可用性的变化来调节。
