Modulation of sodium channel gating

钠通道门控的调制

• 批准号: RGPIN-2018-03920
• 负责人: Ruben, Peter
• 金额: $ 6.12万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746231
• 关键词: Modulation; sodium; channel; gating

Voltage-gated sodium channels (NaVs) are responsible for the initiation and propagation of action potentials in many excitable cells. NaVs undergo a conformational change in response to changes in the electrical field across cell membranes. NaVs consist of a pore-forming and voltage-sensing alpha subunit, and 1 or 2 'auxillary' beta subunits. The structure of the alpha-subunit of NaVs includes four homologous domains with voltage-sensing domains (VSDs) and pore-forming domains (PDs), the latter of which combine to form a gated, water-filled pathway through which sodium ions flow down their electrochemical gradient. The VSDs are made of 4 membrane-spanning alpha-helices (S1-S4). The S4s in the VSD include a high concentration of positively-charged amino acids (AAs), lysines or arginines, in every third position. The positive AAs form salt bridges with negatively-charged AAs in the S2 and S3 segments of the VSDs. Upon depolarization of the electrical field, the VSDs rearrange such that the S4s rotate and translocate in the extracellular direction, making and breaking salt bridges with the negative AAs in S2 and S3. The 'outward' movement of the S4s creates a small current caused caused by the movement of positive charges within the electrical field. In voltage-clamp recordings, this so-called 'gating current' is typically obscured by the much larger ionic current caused by sodium channels moving through the pore. With a sufficiently large population of channels and the ionic current blocked with tetrodotoxin, however, gating currents can be measured and their properties quantified, giving a direct measure of VSD activity under a variety of conditions. NaVs are subject to post-translational modification, including co-association with beta subunits, phosphorylation by protein kinase A, protein kinase C, tyrosine kinase, and calmodulin-dependent protein kinase II, and by calmodulin-mediated calcium binding. The effects of these post-translational modification processes on ionic currents have been well-studied. However, the effects of post-translational modification on gating currents has not yet been described. In the present application, we propose to test the hypotheses that (1) co-association with beta subunits, (2) phosphorylation, and (3) calmodulin-mediated calcium binding alters biophysical properties of NaV gating currents and VSD movements. We will test these hypotheses using NaVs expressed in Xenopus oocytes, recorded using the cut-open oocyte voltage clamp (COVC) and voltage clamp fluorimetry (VCF). COVC allows gating currents to be measured and their biophysical properties quantified. VCF allows direct measurement of individual voltage-sensor translocation. Combined, these measurements permit a detailed analysis of VSD rearrangement and will allow us to answer the question: how is gating in NaVs modulated by post-translational modification?

电压门控钠通道（NaVs）负责许多可兴奋细胞中动作电位的起始和传播。NaV响应于跨细胞膜的电场的变化而经历构象变化。NaV由一个成孔和电压敏感α亚基和1或2个“辅助”β亚基组成。NaV的α-亚基的结构包括具有电压敏感结构域（VSD）和孔形成结构域（PD）的四个同源结构域，后者联合收割机组合以形成门控的水填充通路，钠离子通过该通路沿其电化学梯度流动。VSD由4个跨膜α-螺旋（S1-S4）组成。VSD中的S4在每三个位置包括高浓度的带正电荷的氨基酸（AA）、赖氨酸或赖氨酸。在VSD的S2和S3段中，正AA与带负电荷的AA形成盐桥。在电场去极化时，VSD重新排列，使得S4在细胞外方向上旋转和移位，与S2和S3中的负AA形成和破坏盐桥。S4的“向外”运动产生了由电场内正电荷运动引起的小电流。在电压钳记录中，这种所谓的“门控电流”通常被钠通道通过孔引起的更大的离子电流所掩盖。然而，在足够大的通道群体和用河豚毒素阻断的离子电流的情况下，可以测量门控电流并量化其性质，从而在各种条件下直接测量VSD活性。NaV经历翻译后修饰，包括与β亚基共缔合，通过蛋白激酶A、蛋白激酶C、酪氨酸激酶和钙调蛋白依赖性蛋白激酶II以及通过钙调蛋白介导的钙结合进行磷酸化。这些翻译后修饰过程对离子电流的影响已经得到了很好的研究。然而，翻译后修饰对门控电流的影响尚未被描述。在本申请中，我们提出测试以下假设：（1）与β亚基的共缔合，（2）磷酸化，和（3）钙调蛋白介导的钙结合改变NaV门控电流和VSD运动的生物物理特性。我们将使用爪蟾卵母细胞中表达的NaVs来测试这些假设，使用切开卵母细胞电压钳（COVC）和电压钳荧光测定法（VCF）记录NaVs。COVC允许测量门控电流并量化其生物物理特性。VCF允许直接测量个体电压传感器易位。结合起来，这些测量允许详细分析VSD重排，并将使我们能够回答这个问题：如何门控在NaVs调制的翻译后修饰？