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

了解调节钾电压门控通道（Kv）活性和细胞表面表达的细胞和分子机制至关重要。Kv通道编码40个a亚基，分为12个亚科，代表了神经系统中表达的最多样化的离子通道家族。这种多样性使千伏通道能够独特地影响电气信息处理的几个方面。这些通道在与辅助亚基结合时起作用，这些亚基调节Kv质膜（PM）靶向、细胞内运输、电导和门控（打开或关闭状态）。过去的研究已经确定SIGMA-1R （S1R）是一种内质网（ER）伴侣，是几种Kv a亚基的非典型辅助亚基。然而，参与S1R调控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在确定参与细胞中s1r介导的Kv通道运输的途径和关键成分，并确定S1R-Kv复合物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，我的团队将重点研究S1R在Kv1.2通道贩运中的作用。利用同源表达系统或S1R-缺失细胞，结合elisa细胞检测、共聚焦显微镜和GST-Pull - down分析，研究以下特定目标：轴1:S1R对Kv1.2通道稳定性的影响；自噬中的作用和泛素-蛋白酶体机制。我最近的研究表明，当与S1R共表达时，Kv.12的基础表达会降低。鉴于S1R参与ERAD、UPR和自噬，我们将研究S1R是否在蛋白酶体和自噬途径中增强Kv1.2降解。轴2:S1R对Kv1.2通道细胞表面表达的调控。S1R对若干Kv通道具有配体依赖性和非依赖性。然而，目前尚不清楚这些独特的药物诱导或药物独立事件是如何在细胞内协调的，以及它如何指导s1r介导的通道运输。因此，我计划在S1R激活或失活时监测Kv1.2 PM的表达和运输。轴3:S1R-Kv1.2通道相互作用的表征：配体调节的结合和结合域的定位。在此，我建议使用永生化细胞系和体外GST-Pull - down的过表达试验，以激动剂和拮抗剂特异性的方式检测S1R与Kv1.2的关联。轴4：培养诱导神经元（iNS）作为新的工作模型。我们将建立一个人类初级神经元模型来研究S1R对Kv1.2通道贩运的调控。对NSE研究的意义和预期贡献：该研究项目将解答与S1R基础生物学和S1R- kv通道运输相关的主要未决问题，这些通道使神经元能够严格调节其兴奋性。

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.