Structural Basis of Ion Selectivity in Calciumn Channels

描述：电压门控钙通道是连接 神经细胞中的电信号与细胞内钙信号通路 例如，允许神经细胞释放神经递质或改变其 基因表达。为了完成这些任务，电压门控的钙离子通道在 对动作电位的反应，并只允许钙离子通过 通道的高选择性毛孔进入细胞内部。的故障。 神经元电压门控钙通道对人类健康有严重影响 人类包括遗传性疾病脊髓小脑性共济失调6型，家族性 偏瘫偏头痛和发作性共济失调2型。 拟议研究的目标是了解 通过钙离子通道的选择性离子通量。为了追求这个宏伟的目标，我们 计划实施三个具体目标：(1)确定孔隙的地形 在L类钙通道中；(2)通过测试来定位钙通道门(S) 与状态相关的巯基修饰剂的可及性；以及(3)测量 L型钙离子通道孔道中的静电电位分布。总而言之， 在这些研究中，我们将测量对巯基修饰的可及性 半胱氨酸取代的A1C L型钙通道突变形式的试剂。如果 被取代的半胱氨酸残基的含硫基侧链被暴露 在孔洞的管腔内，然后共价附着一种巯基修饰 试剂可能会导致离子流经孔道受阻。vbl.使用 以得到的持久块为索引，我们将确定哪些残留物 推测的孔隙衬里序列(S5、P-LOOP、S6段)实际上排列在孔隙中。 我们将确定离子导电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/停用 状态依赖的可及性来定位钙通道的门(S)。一个 选择性离子传输的重要参数是本征静电 孔洞中的电势，这将通过测量 不同电荷的巯基改性剂的改性率。总而言之， 实验中，电流块将被测量为电压钳位， 异源表达钙通道。

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.