The roles of the CaVbeta2 subunit and the synaptic protein interaction site in determining Ca2+ channel function in neuroendocrine secretion

钙离子通过电压门控钙离子通道(Cav)流入神经元，触发神经递质和激素的释放，因此这些通道的细胞内靶向对细胞间通讯具有重要意义。CaV通道由一个CaValpha1亚基和四个CaVbeta亚基中的一个组成，这可能有助于将通道输送到轴突终末。在突触释放中最重要的两种钙通道(CaV2.1和Cav2.2)含有一个允许它们与突触蛋白相互作用的肽序列，因此被称为“突触”部位。 我们建议研究CaVbeta2亚单位和同源位点在中枢神经元中的作用，称为大细胞神经分泌细胞(MNCs)，它分泌催产素和加压素激素。释放发生在MNC细胞体和终末，并随着血浆渗透压的增加而增加。我们发现CaVbeta2在MNC末端中含量丰富，提示它可能在Cav的转运中发挥作用。我们还表明，跨国公司表达缺少Synprint位点的CaV2.1的变体，但不表达Cav2.2的等效变体。这些数据可能解释了观察到，Cav2.2，而不是CaV2.1，触发了MNC细胞体中催产素的释放。 我们建议测试1)CaVbeta2亚单位是否在Cav通道的轴突靶向中发挥作用；2)Cav2.2的突触位点与突触蛋白之间的相互作用是否在触发MNC细胞体内催产素的释放中发挥作用。 我们将比较大脑中缺乏CaVbeta2亚单位的转基因小鼠和对照小鼠MNC终端中Cav的表达，并预测其表达将较低。我们进一步预测，这些小鼠将无法正常释放加压素。我们将通过测量暴露在渗透压力下的对照组和基因敲除小鼠的加压素水平和血浆渗透压来测试这一点。我们将通过干扰钙通道和突触蛋白之间的相互作用以及测量单核细胞胞体的催产素释放来测试突触位点在促进单核细胞胞体和终末神经肽分泌中的作用。我们预测，这种相互作用的中断将抑制通过Cav2.2的流入触发释放的能力，这将支持这种相互作用在激素释放中的作用。因此，这些研究将有助于阐明神经元调节激素和神经递质释放的机制。

The flow of Ca2+ into neurons through voltage-gated Ca2+ (CaV) channels triggers the release of neurotransmitters and hormones and the intracellular targeting of these channels is therefore of fundamental importance for intercellular communication. CaV channels are composed of a CaValpha1 subunit and one of four CaVbeta subunits, which may help transport channels to axon terminals. The two types of Ca2+ channels most important in synaptic release (CaV2.1 and CaV2.2) contain a peptide sequence that allows them to interact with synaptic proteins and is thus called the “synprint” site. We propose to study the roles of the CaVbeta2 subunit and the synprint site in central neurons called the magnocellular neurosecretory cells (MNCs), which secrete the hormones oxytocin and vasopressin. Release occurs from both MNC cell bodies and terminals and increases as plasma osmolality increases. We found that the CaVbeta2 is enriched in MNC terminals, suggesting that it might play a role in CaV transport. We have also shown that MNCs express a variant of CaV2.1 that lacks the synprint site, but do not express an equivalent variant of CaV2.2. These data may explain the observation that CaV2.2, but not CaV2.1, triggers release of oxytocin from the MNC cell bodies. We propose to test 1) whether the CaVbeta2 subunit plays a role in axonal targeting of CaV channels and 2) whether the interaction between the synprint site of CaV2.2 and synaptic proteins plays a role in triggering release of oxytocin from MNC cell bodies. We will compare CaV expression in the MNC terminals of transgenic mice that lack the CaVbeta2 subunit in the brain to that of control littermates and predict that the expression will be lower. We further predict that these mice will fail to release vasopressin properly. We will test this by measuring vasopressin levels and plasma osmolality of control and knockout mice exposed to osmotic stress. We will test the role of the synprint site in facilitating neuropeptide secretion in MNC somata and terminals by interfering with the interaction between Ca2+ channels and synaptic proteins and measuring oxytocin release from MNC cell bodies. We predict that the disruption of this interaction will inhibit the ability of influx through CaV2.2 to trigger release, which would support a role for this interaction in hormone release. These studies will therefore help elucidate the mechanisms that allow neurons to regulate the release of hormones and neurotransmitters.