Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca2+ channel
Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca2+ channel
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DOI：
10.1093/jb/mvs054
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发表时间：
2012-08-01
影响因子：
2.7
通讯作者：
Ohtsuka, Toshihisa
中科院分区：
文献类型：
--
作者：
Kiyonaka, Shigeki;Nakajima, Hiroshi;Ohtsuka, Toshihisa
在神经末梢，活性区蛋白CAST/ERC2与其他活性区蛋白ELKS、Bassoon、Piccolo、RIM1和Munc13-1形成蛋白复合物，被认为在神经递质释放中发挥组织和功能作用。然而，CAST/ERC2如何调节Ca2+依赖性神经递质的释放仍然不清楚。在这里，我们展示了CAST与电压依赖性Ca2+通道（VDCCs）的相互作用，这对于调节由去极化诱导的Ca2+内流在活性区触发的神经递质释放是必不可少的。通过生化分析，我们发现CAST与小鼠脑中的VDCC β(4)-亚基共免疫沉淀。下拉实验显示，VDCC β(4)-亚基至少与CAST的N端和c端区域直接相互作用。VDCC α(1)-亚基的II-III连接体也与CAST的c端区相互作用；但与β(4)-亚基相比，其相互作用弱得多。此外，CAST和VDCCs在幼鼠肾细胞中的共表达导致激活的电压依赖性向超极化方向转变。综上所述，这些结果表明CAST与VDCCs形成一种蛋白质复合物，其可能部分通过改变突触前活跃区VDCCs的开放来调节神经递质释放。
In the nerve terminals, the active zone protein CAST/ERC2 forms a protein complex with the other active zone proteins ELKS, Bassoon, Piccolo, RIM1 and Munc13-1, and is thought to play an organizational and functional role in neurotransmitter release. However, it remains obscure how CAST/ERC2 regulates the Ca2+-dependent release of neurotransmitters. Here, we show an interaction of CAST with voltage-dependent Ca2+ channels (VDCCs), which are essential for regulating neurotransmitter release triggered by depolarization-induced Ca2+ influx at the active zone. Using a biochemical assay, we showed that CAST was coimmunoprecipitated with the VDCC beta(4)-subunit from the mouse brain. A pull-down assay revealed that the VDCC beta(4)-subunit interacted directly with at least the N- and C-terminal regions of CAST. The II-III linker of VDCC alpha(1)-subunit also interacted with C-terminal regions of CAST; however, the interaction was much weaker than that of beta(4)-subunit. Furthermore, coexpression of CAST and VDCCs in baby hamster kidney cells caused a shift in the voltage dependence of activation towards the hyperpolarizing direction. Taken together, these results suggest that CAST forms a protein complex with VDCCs, which may regulate neurotransmitter release partly through modifying the opening of VDCCs at the presynaptic active zones.