Center for Neural Science and WCI Center for Functional Connectomics, Korea Institute of Science and Technology (KIST), Seoul, Korea.
Mol Brain. 2013 Dec 9;6:54. doi: 10.1186/1756-6606-6-54.
Glutamate is the major neurotransmitter that mediates a principal form of excitatory synaptic transmission in the brain. From the presynaptic terminals of neurons, glutamate is released upon exocytosis of the glutamate-packaged vesicles. In recent years, astrocytes are also known to release glutamate via various routes to modulate synaptic transmission. In particular, we have characterized a glutamate-permeable Ca2+-activated anion channel encoded by Bestrophin 1 gene (Best1) that is responsible for Ca2+-dependent, channel-mediated glutamate release in astrocyte. Best1 channel contains a large pore that is readily permeable to large molecules such as glutamate and GABA. In those studies we obtained permeability ratio of glutamate to Cl- in heterologously expressed mouse Best1 in HEK293T cells and in endogenously expressed mouse Best1 in cultured astrocytes. However, up to now, glutamate permeability of the native Best1 channel in vivo has not been reported.
In whole-cell recordings of CA1 hippocampal astrocytes, we found that opening of Best1 channel upon activation of a Gq-coupled GPCR, protease-activated receptor 1 (PAR1) generated the anion current carried by glutamate via Ca2+ increase. This Ca2+-evoked glutamate-mediated anion current was unaffected by pretreatment of the inhibitors for a gap junction hemi-channel or Ca2+-activated K+ conductance. This astrocytic anion conductance carried by glutamate was mediated by Best1 channel expression in CA1 hippocampal astrocytes, because Best1 knock-down by shRNA expression eliminated astrocytic glutamate conductance by PAR-1 activation. However, we found that these astrocytes showed a deviation in reversal potential of Best1-mediated current from the predicted value. By performing dual patch recording, we concluded that the deviation of reversal potential is due to incomplete space clamping arising from extremely leaky membrane (input resistance ranging 1-3 MΩ), very low length constant of astrocytic processes, and the localization of Best1 channel in distal microdomains near synapses. Based on the relative shift of reversal potentials by ion substitutions, we estimated the permeability ratio of glutamate and Cl- (Pglutamate/PCl) as 0.53.
Our study shows that Best1, located at the microdomains near the synaptic junctions, has a significantly high permeability to glutamate in vivo, serving as the prominent glutamate-releasing channel in astrocytes, mediating the release of various gliotransmitters in the brain, and playing an important role in modulating synaptic transmission.
谷氨酸是一种主要的神经递质，介导大脑中主要的兴奋性突触传递形式。谷氨酸从神经元的突触前末梢通过谷氨酸包装囊泡的胞吐作用释放。近年来，星形胶质细胞也被发现通过各种途径释放谷氨酸来调节突触传递。特别是，我们已经鉴定了一种由 Bestrophin 1 基因（Best1）编码的谷氨酸通透性钙激活阴离子通道，该通道负责星形胶质细胞中钙依赖性、通道介导的谷氨酸释放。Best1 通道包含一个大的孔，容易通透如谷氨酸和 GABA 等大分子。在那些研究中，我们获得了在 HEK293T 细胞中异源表达的小鼠 Best1 中和在培养的星形胶质细胞中内源性表达的小鼠 Best1 中谷氨酸对 Cl-的通透性比值。然而，到目前为止，体内天然 Best1 通道的谷氨酸通透性尚未报道。
在 CA1 海马星形胶质细胞的全细胞膜片钳记录中，我们发现 Gq 偶联 GPCR 蛋白酶激活受体 1（PAR1）的激活会打开 Best1 通道，从而产生由 Ca2+增加介导的谷氨酸阴离子电流。这种 Ca2+ 诱导的谷氨酸介导的阴离子电流不受间隙连接半通道或 Ca2+ 激活的 K+ 电导抑制剂预处理的影响。这种由谷氨酸介导的星形胶质细胞阴离子电导是由 CA1 海马星形胶质细胞中 Best1 通道的表达介导的，因为 shRNA 表达的 Best1 敲低消除了 PAR-1 激活引起的星形胶质细胞谷氨酸电导。然而，我们发现这些星形胶质细胞的 Best1 介导电流的反转电位偏离了预测值。通过进行双膜片钳记录，我们得出结论，反转电位的偏差是由于源自极其渗漏的膜（输入电阻范围为 1-3 MΩ）的不完全空间钳位、星形胶质细胞突起的极低长度常数以及 Best1 通道在突触附近的远端微域中的定位引起的。基于离子取代引起的反转电位的相对偏移，我们估计谷氨酸和 Cl-的通透性比值（Pglutamate/PCl）为 0.53。
我们的研究表明，位于突触结附近的微域中的 Best1 对体内谷氨酸具有显著的高通透性，作为星形胶质细胞中主要的谷氨酸释放通道，介导大脑中各种神经递质的释放，并在调节突触传递中发挥重要作用。
