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

描述（由申请人提供）：n型Ca通道与P/ q型通道一起，在神经系统的化学突触传递中起着核心作用。由于这些Ca2+通道决定了Ca2+在神经末梢内流的数量、时间和位置，Ca2+通道生物物理特性的变化，特别是它们的失活动力学，可以深刻影响诱发神经递质释放的时间动力学。尽管最近取得了进展，但我们对Ca2+通道失活的分子机制以及神经元中信号蛋白对其的调节的理解仍然存在重大差距。本研究旨在了解14-3-3蛋白调控n型Ca2+通道的细胞和分子机制，14-3-3蛋白是一个参与多种细胞过程的富脑蛋白家族。在我们的初步研究中，我们不仅发现并表征了n型Ca2+通道与14-3-3之间新的蛋白-蛋白相互作用，而且还确定了14-3-3对n型Ca2+通道失活特性的深刻调节。此外，我们还发现通过拮抗突触前神经元的14-3-3结合，短期突触可塑性发生了显著变化。在本应用中，我们将以这些发现为基础，利用分子、生化和电生理技术的结合，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依赖的n型Ca2+通道失活的调节机制。具体来说，我们将研究14-3-3是否通过与n型Ca2+通道的结合来调节n型Ca2+通道的失活特性。(2)确定14-3-3依赖性调节短期突触可塑性的机制。具体来说，我们将研究14-3-3是否通过调节n型Ca2+通道失活来调节短期可塑性。(3)确定14-3-3与神经元内n型Ca2+通道的动态相互作用。具体来说，我们将研究这种蛋白复合物的形成是否通过反复出现的突触前活动，通过增强camkii依赖性的n型Ca2+通道磷酸化来促进。总之，这些研究将提供n型Ca2+通道调控的新机制，并有助于理解14-3-3在神经系统中的功能。

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.