CELLULAR MECHANISMS INVOLVED IN VOLTAGE-GATED POTASSIUM CHANNEL REGULATION: FOCUS ON SIGMA-1R DRIVEN PATHWAYS

了解钾电压门控通道(Kv)活性和细胞表面表达的细胞和分子机制是至关重要的。Kv通道编码40个a亚基，分为12个亚家族，是神经系统中表达的最多样化的离子通道家族。这种多样性使Kv通道能够独一无二地影响电信息处理的几个方面。当与辅助亚基结合时，这些通道是有功能的，辅助亚基调节Kv质膜(PM)靶向、细胞内转运、电导和门控(打开或关闭状态)。以往的研究发现Sigma-1R(S1R)是内质网(ER)伴侣蛋白，是几个Kvα亚基的非典型辅助亚基。然而，S1R调节Kv通道的分子机制仍然知之甚少。因此，我的发现资助计划的长期目标将集中于确定参与S1R介导的Kv通道在细胞内运输的途径和关键成分，并确定S1R-Kv复合体在神经元兴奋性中的作用，这是正常大脑功能的基本机制。在接下来的五年里，我的团队将专注于研究S1R在Kv1.2渠道贩运中的作用。利用同源表达系统或S1R缺失的细胞，结合基于ELISA细胞的分析、共聚焦显微镜和GST-下拉分析，将研究以下特定目标：轴1：S1R对Kv1.2通道稳定性的影响：在自噬中的作用和泛素-蛋白酶体机制。我最近的工作表明，当Kv.12与S1R共表达时，Kv.12的基础表达减少。鉴于S1R参与ERAD、UPR和自噬，我们将研究S1R是否沿着蛋白酶体和自噬途径促进Kv1.2的降解。轴2：S1R对Kv1.2通道细胞表面表达的调节。已证明S1R对几个Kv通道具有配体依赖和独立作用。然而，目前尚不清楚这些独特的药物诱导或药物非依赖性事件是如何在细胞内编排的，以及它是如何指导S1R介导的通道贩运的。因此，我计划监测Kv1.2 PM在S1R激活或失活时的表达和运输。轴3：S1R-Kv1.2通道相互作用的特征：配体调节的结合和结合结构域的映射。因此，我建议在永生化细胞系中用激动剂和拮抗剂特异性的方式检测S1R与Kv1.2的相关性，并在体外用GST-Dull-down检测S1R的过度表达。轴4：发展诱导神经元(INS)作为一种新的工作模式。我们将建立一个人类原代神经元模型来研究S1R对Kv1.2通道运输的调节。对NSE研究的意义和预期贡献：该研究计划将为与S1R基础生物学和S1R-Kv通道运输相关的主要悬而未决的问题提供答案，使神经元能够严格控制其兴奋性。

Understanding the cellular and molecular mechanisms modulating potassium voltage-gated channel (Kv) activity and cell surface expression is of the utmost importance. Encoding 40 a-subunits divided in twelve subfamilies, the Kv channels represent the most diverse family of ion channels expressed in the nervous system. This diversity allows Kv channels to uniquely influence several aspects of electrical information processing. These channels are functionnal when bound to auxiliary subunits, which modulate the Kv plasma membrane (PM) targeting, intracellular trafficking, conductance, and gating (open or closed state). Past studies have identified SIGMA-1R (S1R), an endoplasmic reticulum (ER) chaperone, as an atypical auxiliary subunit for several Kv a-subunits. However, the molecular mechanisms involved in S1R regulation of Kv channels is still poorly understood. Therefore, the long term objective of my Discovery grant program will focus on identifying the pathways and key components engaged in S1R-mediated Kv channel trafficking in cells and to define the S1R-Kv complexes' role in neuronal excitability, a fundamental mechanism for proper brain functions. For the next five years, my team will focus on studying the role of S1R in Kv1.2 channel trafficking. Using homologous expression system or S1R-depleted cells combined with ELISA-cell based assays, confocal microscopy and GST-Pull down analyses, the following specific objectives will be investigated: Axis 1:  Impact of S1R on Kv1.2 channel stability: role in autophagy and the ubiquitin-proteasome mechanisms. My most recent work exposed that Kv.12 basal expression is reduced when co-expressed with S1R. Given that S1R is involved in ERAD, UPR, and autophagy, we will investigate if S1R enhances Kv1.2 degradation along the proteasomal and autophagy pathways. Axis 2: S1R regulation of Kv1.2 channel Cell Surface Expression. S1R has been shown to have ligand-dependent and independent effects on several Kv channels. However, it remains unclear how these distinctive drug-inducible or drug-independent events are orchestrated within cells and how it is guiding S1R-mediated channels trafficking. Hence, I plan to monitor Kv1.2 PM expression and trafficking upon S1R activation or inactivation. Axis 3: Characterization of S1R-Kv1.2 channel interaction: ligand-regulated association and mapping of the binding domain.  Herein, I suggest examining S1R association with Kv1.2 in an agonist and antagonist-specific manner using overexpression assays in immortalized cell lines and in vitro GST-Pull down. Axis 4: Develop induced Neurons (iNS) as a new working model. We will develop a human primary neuronal model to study S1R regulation of Kv1.2 channel trafficking. Significance and expected contributions to NSE research: This research program will provide answers to major pending questions related to S1R fundamental biology and S1R-Kv channels trafficking that enable neurons to tightly regulate their excitability.