Calcium Channel Gating: It Matters How You Splice It.

描述(申请人提供)：该项目的长期目标是了解电压门控钙离子通道如何感知和解码调节神经递质释放的电子和分子信号。钙通道是一种大的(约370 kDa)异多聚体蛋白，由α1、α2β和β亚基组成，它们协同工作以控制对给定刺激进入神经元的钙离子的量。这些亚基形成钙通道孔(4个大的跨膜同源结构域)，由至少10个基因编码，根据结构、电生理和药理学差异，可分为三个主要亚群：Cav1、Cav2和Cav3。Cav1和Cav2是高压激活通道，而Cav3通道在较负的膜电位下激活。Cav1基因表达具有L型(长持续)电生理特征的通道，Cav2基因表达P/Q、N和R型(中间持续)通道，Cav3基因表达T型(瞬变)通道。α1亚基是几类治疗药物的靶点，包括抗心律失常药物(地尔硫卓，L型拮抗剂)和止痛药(齐康肽，海洋蜗牛的N型拮抗剂)，以及一系列多肽蜘蛛毒素(例如，Aga IVA，P型拮抗剂)。Cav2基因主要在突触中表达，细胞内的β亚基由4个不同的基因编码，在同源结构域连接序列的特定结合位置与α1亚基相互作用。β亚基调节钙通道的表达水平，以及钙通道激活和失活的电压依赖性和动力学。我们的初步研究表明，Beta4亚基N-末端的选择性剪接对钙通道门控具有α1亚基亚型特异性的影响。他们还表明，剪接影响通道药理学(改变了对omegaCgTx GVIA的敏感性)和α1亚单位对重复刺激的反应性。因此，了解Beta4选择性剪接所带来的事件的分子细节对于开发止痛药以及进一步了解电压门控钙通道在突触可塑性中所起的作用是至关重要的。为此，我们通过使用结构基因组学中的简单方法获得的最显著的初步结果是发现Beta4亚基和突触支架(Maguk)蛋白PSD-95是从一个共同的祖先进化而来的。这两种蛋白质具有非常相似的预测二级结构，随着PSD-95的晶体结构现已可用，现在可以进行许多Beta4亚基三级结构的预测。这项应用的目的是利用先进的核磁共振技术证实我们的三级结构预测，并确定PSD-95分子间和分子内的良好相互作用是否在Beta4亚基中保守。我们的假设是，Beta4亚基作为多种蛋白质的多模块对接位置，包括钙调蛋白、激酶锚定蛋白和pdz结构域，并作为导向器，将分子信号从细胞内传递到alpha1亚基的门控机制。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand how voltage-gated Ca2+ channels sense and decode electrical and molecular signals that regulate neurotransmitter release. Ca2+ channels are large (about 370 kDa) heteromultimeric proteins composed of alpha1, alpha2delta, and beta subunits that work in concert to control the amount of Ca2+ that enters a neuron in response to a given stimulus. The subunits form the Ca2+ channel pore (4 large transmembrane homology domains) and are encoded by at least ten genes that, based on structural, electrophysiological, and pharmacological differences, can be divided into three major sub-groups, Cav1, Cav2 and Cav3. Cav1 and Cav2 are high-voltage activating channels, whereas Cav3 channels activate at more negative membrane potentials. Cav1 genes express channels with L-type (long lasting) electrophysiological characteristics, Cav2 genes express P/Q, N, and R-type (intermediate lasting) channels, and Cav3 genes express T-type (transient) channels. The alpha1 subunits serve as targets for several classes of therapeutic agents, including antiarrhythmics (diltiazem, L-type antagonist) and analgesics (ziconotide, N-type antagonist from a marine snail), and for a host of peptide spider toxins (e.g., Aga IVA, P-type antagonist). Cav2 genes, which will be studied in this proposal, are expressed principally at synapses.The intracellular beta subunits, encoded by 4 distinct genes, interact with the alpha1 subunit at specific binding sites on between-homology-domain linker sequences. The beta subunits modulate Ca2+ channel expression levels, as well as the voltage dependence and kinetics of Ca2+ channel activation and inactivation. Our preliminary studies show that alternative splicing of the N-terminus of the beta4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating. They also show that splicing affects channel pharmacology (altered sensitivity to omegaCgTx GVIA) and responsiveness of alpha1 subunits to repetitive stimuli. Thus, understanding the molecular details of the events brought about by beta4 alternative splicing is essential for the development of analgesic drugs, and for furthering our understanding of the role that voltage-gated Ca2+ channels play in synaptic plasticity. To this end, our most remarkable preliminary result, obtained by using simple methods in structural genomics, is the discovery that the beta4 subunit and the synaptic scaffolding (MAGUK) protein, PSD-95, have evolved from a common ancestor. The two proteins share very similar predicted secondary structure, and with the crystal structure of PSD-95 now available, a number of beta4 subunit tertiary structure predictions can now be made. The objectives of this application are to confirm, using advanced NMR techniques, our tertiary structure predictions and to determine whether the well-characterized inter- and intramolecular interactions of PSD-95 have been conserved in beta4 subunits. Our hypothesis is that the beta4 subunit acts as a multi-modular docking site for a myriad of proteins, including calmodulin, kinase anchoring proteins, and PDZ domains, and serves as a director, transmitting molecular signals from inside the cell to the gating machinery of alpha1 subunits