Subconductance behavior in T-type calcium channels

说明(申请人提供)：电压门控钙(Ca)通道有两个主要作用，一个是通过传递电流从而影响膜电位的生电作用，另一个是调节作用，因为它们对重要的信号分子钙具有选择性的通透性。全球细胞内和局部钙浓度的变化导致可溶性α依赖蛋白和通道的激活，从而控制不同的细胞过程，包括膜兴奋性、细胞死亡、生长、囊泡释放和突触可塑性。因此，钙的调节是关键的，而这种调节的破坏是病理生理的。T型钙通道是电压门控钙通道家族中的一员，在大脑中广泛表达。它们既有电的作用，也有调节作用，因为它们有助于低阈值钙峰、起搏、反弹爆发放电，以及在静息膜电位附近产生稳定的钙内流和低幅度的钙振荡。它们与几种神经系统疾病有关，包括但不限于失神发作、神经源性疼痛、睡眠障碍和共济失调。这项研究旨在了解这些通道的渗透，以更好地了解它们如何参与细胞生理学和病理生理学。具体地说，将使用单通道膜片钳技术来检测这些通道中的亚电导行为，以提供通过这些通道的钙通量的定量测量。首先，将确定Cav 3.1的电导状态的数量以及每个状态的电压依赖性和动力学。Cav 3.1是一种在大脑中广泛表达的T通道亚型。其次，将测量所有电导水平的条件概率，以检验次导电态是由门控过程中气孔的部分打开引起的假设。最后，由于T通道被认为具有依赖于其他离子的复杂的渗透“规则”，因此在存在不同的阳离子、钙、钡和一价阳离子的情况下，将测量每个电导态的相对幅度和动力学。在第二个目标中，将评估CAV 3.1和CAV 3.2之间的数量差异。这两种异构体在大脑中表现出互补的表达模式，并且在发育过程中差异表达。确定这两种异构体之间的异同将有助于理解每一种异构体如何对它们所参与的细胞生理做出贡献。在此获得的数据将有助于理解这些通道在各种神经疾病中的作用，并有可能开发选择性靶向这些通道并保留其他电压门控通道的药物。

DESCRIPTION (provided by applicant): Voltage-gated calcium (Ca) channels serve two major roles, an electrogenic role by passing current and thereby affecting membrane potential and a regulatory role because they are selectively permeable to Ca, an important signaling molecule. Changes in global intracellular and local Ca concentrations lead to the activation of soluble a dependent proteins and channels and thereby control diverse cell processes including membrane excitability, cell death, growth, vesicular release, and synaptic plasticity. Regulation of Ca is, therefore, critical and disruption of this regulation is pathophysiological. T-type Ca channels, one family of voltage-gated Ca channels, are widely expressed in the brain. They serve both an electrogenic and regulatory role in that they contribute to lowthreshold Ca spikes, pacemaking, rebound burst firing as well as producing a steady Ca influx near the resting membrane potential and low amplitude Ca oscillations. They have been implicated in several neurological disorders including but not limited to absence seizures, neurogenic pain, sleep disorders, and ataxia. This studys aimed at understanding permeation through these channels in an effort to better understand how they can participate in cellular physiologies and pathophysiologies. Specifically subconductance behavior in these channels will be examined using single channel patch clamp techniques to provide a quantitative measure of Ca flux through these channels. First the number of conductance states and the voltage dependence and kinetics of each for CaV 3.1, a T-channel isoform broadly expressed in the brain, will be determined. Second, conditional probabilities for all conductance levels will be measured to test the hypothesis that subconductance states arise From partial opening of the pore during gating. Lastly because T channels are thought to have complex permeation "rules" that are dependent on other ions, the relative amplitude and kinetics of each conductance state will be measured in both the presence of different permeant cations, Ca, barium, and monovalent cations. In the second aim quantitative differences between CaV 3.1 and CaV 3.2 will be assessed. These two isoforms display complementary expression patterns in the brain and are differentially expressed during development. Identifying similarities and differences between these two isoforms will aid in understanding how each contributes to the cellular physiologies in which they participate. Data obtained here will be helpful in understanding the role of these channels in the variety of neurological diseases in which they play a role and potentially in developing drugs that selectivity target these channels and spare other voltage-gated channels.