Sakane Ayuko, Miyoshi Jun, Takai Yoshimi, Sasaki Takuya
Department of Biochemistry, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
Methods Enzymol. 2008;438:131-9. doi: 10.1016/S0076-6879(07)38009-9.
Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Recently mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GTPase-activating protein were found to cause Warburg Micro syndrome and Martsolf syndrome, respectively, both of which exhibit mental retardation. We have found that loss of p130 in mice results in inhibition of Ca2+-dependent glutamate release from cerebrocortical synaptosomes and alters short-term plasticity in the hippocampal CA1 region, probably through the accumulation of the GTP-bound form of Rab3A. Here, we describe the procedures for the measurement of the GTP-bound pool of Rab3A with pull-down assay using mouse brains and the biochemical method for the measurement of glutamate release from mouse synaptosomes.
越来越多的证据表明，Rab3A在神经递质释放和突触可塑性中起关键作用。最近发现，Rab3 GTP酶激活蛋白的催化亚基p130和非催化亚基p150中的突变分别导致沃伯格微综合征和马茨洛夫综合征，这两种综合征均表现出智力迟钝。我们发现，小鼠中p130的缺失会抑制大脑皮质突触体中Ca2+依赖性谷氨酸的释放，并改变海马CA1区的短期可塑性，这可能是通过Rab3A的GTP结合形式的积累实现的。在此，我们描述了使用小鼠大脑通过下拉分析法测量Rab3A的GTP结合池的程序，以及测量小鼠突触体中谷氨酸释放的生化方法。
