Structural Basis of Ion Selectivity in Calciumn Channels

描述：电压门控Ca 2+通道是连接 神经细胞的电信号和细胞内Ca 2+信号通路 例如，它允许神经细胞释放神经递质或改变它们的 基因表达。为了完成这些任务，电压门控Ca 2+通道打开， 对动作电位的反应，并仅允许Ca 2+通过 通道的高度选择性孔进入细胞内部。故障 神经元电压门控Ca 2+通道具有严重的健康后果， 人类，包括遗传性疾病脊髓小脑共济失调6型，家族性 偏瘫性偏头痛和发作性共济失调2型。 本研究的目的是了解 通过Ca 2+通道的选择性离子通量。为了实现这一宏伟目标，我们 计划开展三个具体目标：（1）确定孔隙的形貌 在L型Ca 2+通道中;（2）通过测试 巯基修饰剂的状态依赖性可及性;和（3）测量 L型Ca 2+通道孔中的静电势分布。在所有 在这些研究中，我们将测量巯基修饰的可及性， a1 c L型Ca 2+通道的半胱氨酸取代突变形式的药剂。如果 暴露出取代的半胱氨酸残基的带有巯基的侧链 在孔的内腔中，然后共价连接巯基修饰的 试剂可能导致渗透离子流通过孔的阻塞。使用 作为一个索引，我们将确定哪些残留的 推定的孔衬序列（S5、P环、S6片段）实际上衬在孔上。 我们将确定离子传导孔的几个部分的尺寸 （外部和内部前庭，离子选择性过滤器）使用 各种尺寸的巯基改性剂。我们将使用打开/关闭/未激活 状态依赖的可接近性，以定位Ca 2+通道的门。一个 选择性离子迁移的重要参数是固有的静电 孔中的电位，这将通过测量 不同电荷的巯基改性剂的改性速率。在所有 实验中，将测得的电流块进行电压箝位， 异源表达的Ca 2+通道。

DESCRIPTION: Voltage-gated Ca2+ channels are the principal link between electrical signals in nerve cells and intracellular Ca2+ signaling pathways that allow nerve cells to, for example, release neurotransmitter or alter their gene expression. To accomplish these tasks, voltage-gated Ca2+ channels open in response to an action potential and allow exclusively Ca2+ to travel through the channel's highly selective pore into the cellular interior. Malfunction of neuronal voltage-gated Ca2+ channels has serious health consequences for humans, including the genetic diseases spinocerebellar ataxia type 6, familial hemiplegic migraine, and episodic ataxia type-2. The goal of the proposed research is to understand the structural basis of selective ion flux through Ca2+ channels. In pursuit of this broad goal, we plan to carry out three Specific Aims: (1) determine the topography of the pore in an L-type Ca2+ channel; (2) localize Ca2+ channel gate(s) by testing for state-dependent accessibility of sulfhydryl-modifiers; and (3) measure the electrostatic potential profile in the pore of an L-type Ca2+ channel. In all of these studies we will measure the accessibility to sulfhydryl-modifying agents of cysteine-substituted mutant forms of the a1c L-type Ca2+ channel. If the sulfhydryl-bearing side chain of a substituted cysteine residue is exposed in the lumen of the pore, then covalent attachment of a sulfhydryl-modifying reagent may result in obstruction of permeant ion flow through the pore. Using the resulting persistent block as an index, we will determine which residues of putative pore-lining sequences (S5, P-loop, S6 segment) in fact line the pore. We will determine the dimensions of several parts of the ion-conducting pore (external and internal vestibules, ion selectivity filter) using sulfhydryl-modifiers of various sizes. We will use open/closed/inactivated state-dependent accessibility to localize the gate(s) of the Ca2+ channel. An important parameter of selective ion transport is the intrinsic electrostatic potential in the pore, and this will be determined from measurements of modification rate for differently charged sulfhydryl modifiers. In all experiments, block of current will be measured for voltage-clamped, heterologously expressed Ca2+ channels.