Resurgent Na current: regulation and reconstitution in cultured neurons

复苏的钠电流：培养神经元的调节和重建

• 批准号: 7454145
• 负责人: TERESA K AMAN
• 金额: $ 2.76万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7454145
• 关键词: Affect; Area; Basal Ganglia; Biological Assay; Brain; Brain region; Cells; Cerebellum; Cytoplasmic Tail; Disease; Disease model; Down-Regulation; Fire - disasters; Frequencies; Functional disorder; Goals; Hippocampus (Brain); Huntington Disease; Kinetics; Knowledge; Maintenance; Modeling; Molecular; Motor; Mus; Neuraxis; Neurons; Pattern; Peptides; Protein Subunits; Proteins; Purkinje Cells; Pyramidal Cells; Regulation; Symptoms; System; Testing; Up-Regulation; Western Blotting; base; bean; hippocampal pyramidal neuron; mouse model; particle; reconstitution; research study; sodium channel proteins; voltage; voltage clamp

DESCRIPTION (provided by applicant): Open-channel block and unblock of voltage-gated Na channels, which results in resurgent Na current, is a specialized form of Na channel inactivation in many neurons. It increases channel availability and high frequency firing in cerebellar Purkinje cells. Upregulation or downregulation of the open channel blocking protein may change neuronal spiking, possibly providing a target for treatment of disease states that result from pathological firing patterns. The molecular mechanisms of this form of inactivation, however, have not been fully described. Knowledge of these mechanisms will increase understanding in the maintenance of activity patterns in cells expressing the specialized form of inactivation and may allow directed manipulation of activity in any neuron. Therefore, the major goals of this proposal are to define the mechanisms of open- channel block, specifically the identity of the blocker and whether it can be modulated in disease states. Specific aim one is to test the sufficiency of the Na channel beta4 subunit to produce block. Hippocampal CA3 pyramidal cells express the same alpha-subunits as Purkinje cells but do not have specialized inactivation kinetics, suggesting they do not contain the blocker (Raman and Bean, 1997). Based on the observation that the beta4 cytoplasmic tail peptide can mimic endogenous block (Grieco et al., 2005), expression of beta4 in CAS pyramidal neurons may be sufficient to produce resurgent current. To test this idea, the beta4 protein will be expressed in cultured CAS pyramidal cells. We will test for resurgent current by pulling nucleated patches from these cells and recording in voltage-clamp. The second aim of these experiments is based upon the observation that Na channel beta4 is significantly decreased in the cortex and basal ganglia in a model of Huntington's disease concurrently with symptom onset (Oyama et al., 2006). Thus, we will use these mice as a functional knockdown of beta4 to test if this protein can function as the blocking particle that produces resurgent current. We will first determine whether beta4 is reduced in cerebellar Purkinje cells using Western blotting and if so, acutely dissociate Purkinje cells and test for resurgent current by recording Na currents in voltage-clamp. We will also test whether some motor dysfunction of the disease can be attributed to the changes in resurgent current. Voltage-gated sodium channels are proteins responsible for the excitability of neurons in the central nervous system. Excitability is strongly limited by inactivation, a conformational change of these proteins. This project involves studying the molecular mechanisms of a form of Na channel inactivation, which could aid in the study of normal or pathological neuronal activity.

描述(申请人提供)：开放通道阻断和解除阻断电压门控钠通道，导致钠电流复苏，是许多神经元中钠通道失活的一种特殊形式。它增加了小脑浦肯野细胞的通道可用性和高频放电。开放通道阻断蛋白的上调或下调可能改变神经元的尖峰，可能为治疗由病理性放电模式引起的疾病状态提供靶点。然而，这种形式的失活的分子机制还没有完全被描述。对这些机制的了解将增加对维持细胞中表达特殊形式的失活的活动模式的理解，并可能允许直接操纵任何神经元的活动。因此，这项提案的主要目标是确定开放通道阻断的机制，特别是阻滞剂的身份，以及它是否可以在疾病状态下进行调节。具体目的之一是检测钠通道β4亚基是否足以产生阻断剂。海马区CA3锥体细胞表达与浦肯野细胞相同的阿尔法亚单位，但没有特殊的失活动力学，这表明它们不包含阻滞剂(Raman和Bean，1997)。根据Beta4胞浆尾肽可以模拟内源性阻断的观察(Grieco等人，2005年)，在CAS锥体神经元中Beta4的表达可能足以产生复活电流。为了验证这一想法，将在培养的CAS锥体细胞中表达Beta4蛋白。我们将通过从这些细胞中拉出有核的贴片并在电压钳中记录来测试复苏的电流。这些实验的第二个目的是基于观察到在亨廷顿病症状出现的同时，大脑皮质和基底节中Na通道Beta4显著减少(Oyama等人，2006年)。因此，我们将使用这些小鼠作为Beta4的功能性敲除，以测试这种蛋白质是否可以作为产生复活电流的阻断粒子发挥作用。我们将首先用Western blotting确定小脑浦肯野细胞中的Beta4是否减少，如果是，则急性分离浦肯野细胞，并通过在电压钳上记录钠电流来测试复活电流。我们还将测试一些运动功能障碍的疾病是否可以归因于复苏电流的变化。电压门控钠通道是负责中枢神经系统神经元兴奋性的蛋白质。兴奋性受到失活的强烈限制，失活是这些蛋白质的构象变化。这个项目涉及研究一种形式的钠通道失活的分子机制，这可能有助于研究正常或病理的神经元活动。