Resurgent Na current: regulation and reconstitution in cultured neurons

描述（由申请人提供）：电压门控Na通道的开放通道阻断和解除阻断（导致Na电流复苏）是许多神经元中Na通道失活的一种特殊形式。它增加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以改变神经元尖峰，可能提供用于治疗由病理性放电模式引起的疾病状态的靶标。然而，这种失活形式的分子机制尚未完全描述。这些机制的知识将增加对表达失活的专门形式的细胞中的活动模式的维持的理解，并且可以允许对任何神经元中的活动进行定向操纵。因此，该提案的主要目标是定义开放通道阻滞的机制，特别是阻滞剂的身份以及它是否可以在疾病状态下调节。具体目标一是检测Na通道β 4亚基产生阻滞的充分性。海马CA3锥体细胞表达与浦肯野细胞相同的α亚单位，但不具有专门的失活动力学，表明它们不含阻滞剂（拉曼和Bean，1997）。基于β 4胞质尾肽可以模拟内源性阻断的观察（Grieco et al.，2005），β 4在CAS锥体神经元中的表达可能足以产生复苏电流。为了验证这个想法，β 4蛋白将在培养的CAS锥体细胞中表达。我们将通过从这些细胞中拉出有核斑片并在电压钳中记录来测试复活电流。这些实验的第二个目的是基于以下观察结果：在症状发作的同时，在亨廷顿病模型中，Na通道β 4在皮质和基底神经节中显著降低（Oyama等人，2006年）。因此，我们将使用这些小鼠作为β 4的功能性敲低，以测试这种蛋白质是否可以作为产生复活电流的阻断颗粒。我们将首先使用蛋白质印迹法确定小脑浦肯野细胞中β 4是否减少，如果是，则急性分离浦肯野细胞并通过记录电压钳中的Na电流来测试复苏电流。我们也将测试是否某些运动功能障碍的疾病可以归因于复活电流的变化。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。该项目涉及研究Na通道失活形式的分子机制，这可能有助于研究正常或病理性神经元活动。

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.