Modulation of sodium channel gating

电压门控钠通道(NAV)在许多可兴奋细胞中负责动作电位的起始和传播。NAV会随着细胞膜上电场的变化而发生构象变化。NAV由一个成孔和电压敏感的α亚基和1个或2个‘辅助’β亚基组成。NAWS的α-亚基的结构包括四个同源结构域，包括电压敏感结构域(VSD)和造孔结构域(PDS)，后者结合形成一条门控的充满水的通道，钠离子通过该通道向下流动。VSD由4个跨膜的α-螺旋(S1-S4)组成。VSD中的S4在每三个位置都含有高浓度的正电荷氨基酸(AAs)，即赖氨酸或精氨酸。在VSD的S2和S3节段，正的AA与带负电荷的AA形成盐桥。当电场去极化时，VSD重新排列，使S4在细胞外方向旋转和移位，在S2和S3中与负的AAs建立和打破盐桥。S4的“向外”运动产生了一个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”通常会被钠离子通道通过孔洞时产生的大得多的离子电流所掩盖。然而，有了足够多的通道和被河豚毒素阻断的离子电流，就可以测量门控电流并量化它们的特性，从而直接测量VSD在各种条件下的活性。NAVs受到翻译后修饰的影响，包括与β亚基的共结合，蛋白激酶A、蛋白激酶C、酪氨酸激酶和钙调蛋白依赖的蛋白激酶II的磷酸化，以及钙调蛋白介导的钙结合。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。在目前的应用中，我们建议检验以下假设：(1)与β亚基共结合，(2)磷酸化，(3)钙调素介导的钙结合改变NAV门控电流和VSD运动的生物物理性质。我们将使用非洲爪哇卵母细胞中表达的NAV来验证这些假设，这些NAV使用切开卵母细胞电压钳(COVC)和电压钳荧光法(VCF)记录。COVC允许测量门控电流并量化其生物物理特性。VCF允许直接测量单个电压传感器的移位。这些测量结合在一起，可以对室间隔缺损的重排进行详细的分析，并使我们能够回答这个问题：NAV中的门控是如何受到翻译后修饰的调节的？

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?