Functions of the N-type Ca Channel/14-3-3 Interaction

描述(由申请人提供)：N型钙通道和P/Q型钙通道在神经系统的化学突触传递中起着核心作用。由于这些钙通道决定了神经末梢钙内流的数量、时间和位置，钙通道生物物理性质的改变，特别是其失活动力学，可以深刻地影响诱发神经递质释放的时间动力学。尽管最近取得了一些进展，但我们对钙通道失活的分子机制以及神经元中信号蛋白对其调控的了解仍存在重大差距。这一建议试图了解14-3-3蛋白调节N型钙通道的细胞和分子机制，14-3-3蛋白是一类富含大脑的蛋白质，参与多种细胞过程。在我们的初步研究中，我们不仅发现并表征了N型钙通道与14-3-3之间新的蛋白质-蛋白质相互作用，而且还确定了14-3-3对N型钙通道失活特性的深刻调控。此外，我们通过拮抗突触前神经元中的14-3-3结合，揭示了突触的短期可塑性的显著变化。在这一应用中，我们将在这些发现的基础上，结合分子、生化和电生理技术进一步研究这种调节复合体的功能和机制。我们的具体目标是：(1)确定14-3-3依赖调控N型钙通道失活的机制。具体地说，我们将研究14-3-3是否通过与N型钙通道结合来调节N型钙通道的失活特性。(2)确定14-3-3依赖调节突触短时可塑性的机制。具体地说，我们将研究14-3-3是否通过调节N型钙通道失活来调节短期可塑性。(3)测定神经元内14-3-3和N型钙通道的动态相互作用。具体地说，我们将研究这种蛋白复合体的形成是否通过促进依赖于CaMKII的N型钙通道的磷酸化而被反复的突触前活动促进。这些研究将为N型钙通道的调控提供新的机制，并有助于理解14-3-3‘S在神经系统中的作用。

DESCRIPTION (provided by applicant): The N-type Ca channel, along with the P/Q-type, plays a central role in chemical synaptic transmissions in the nervous system. As these Ca2+ channels determine the amount, timing and location of Ca2+ influx at nerve terminals, changes in Ca2+ channel biophysical properties, especially their inactivation kinetics, can profoundly affect the temporal dynamics of evoked neurotransmitter release. Despite recent advances, there are still major gaps in our understanding of molecular mechanism for Ca2+ channel inactivation, as well as their regulations by signally proteins in neurons. This proposal seeks to understand the cellular and molecular mechanisms of regulation of N-type Ca2+ channels by 14-3-3 proteins, a family of brain-rich proteins that participate in multiple cellular processes. In our preliminary studies, we not only discovered and characterized the novel protein-protein interaction between the N-type Ca2+ channel and 14-3-3, but also determined a profound modulation of inactivation properties of N-type Ca2+ channels by 14-3-3. Furthermore, we revealed a significant change in short-term synaptic plasticity by antagonizing 14-3-3 binding in the presynaptic neuron. In this application, we will build on these findings and further investigate the function and mechanism of this regulatory complex using a combination of molecular, biochemical and electrophysiological techniques. Our specific aims are: (1) Determine the mechanism underlying 14-3-3-dependent modulation of N-type Ca2+ channel inactivation. Specifically, we will investigate whether 14-3-3 modulates inactivation properties of N-type Ca2+ channels through its binding to the channel. (2) Determine the mechanism underlying 14-3-3- dependent modulation of short-term synaptic plasticity. Specifically, we will investigate whether 14-3-3 regulates short-term plasticity by modulating N-type Ca2+ channel inactivation. (3) Determine dynamic interactions between 14-3-3 and N-type Ca2+ channels in neurons. Specifically, we will investigate whether formation of this protein complex is promoted by recurring presynaptic activity, via enhancing CaMKII-dependent phosphorylation of the N-type Ca2+ channel. Together, these studies will provide a novel mechanism for regulation of N-type Ca2+ channels and help to understand 14-3-3's functions in the nervous system.