Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins
Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins
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DOI：
10.1073/pnas.1106811108
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发表时间：
2011-07-26
影响因子：
11.1
通讯作者：
Minor, Daniel L., Jr.
中科院分区：
文献类型：
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作者：
Shaya, David;Kreir, Mohamed;Minor, Daniel L., Jr.
许多电压门控离子通道 (VGIC) 超家族成员包含六次跨膜片段，其中前四个形成电压传感域 (VSD)，后两个形成孔域 (PD)。对 VGIC 超家族钾通道的研究以及仅电压传感器蛋白的鉴定表明 VSD 和 PD 可以独立折叠。这种跨膜模块化是否对于其他 VGIC 超家族成员来说是常见的，尚未得到测试。在这里，我们通过蛋白质解剖表明，Silicibacter pomeroyi 电压门控钠通道 (Na(V)Sp1) PD 形成一个独立的离子选择性孔 (Na(V)Sp1p)，该孔是四聚体、α 螺旋，并且在重构为脂质双层时形成功能性钠选择性通道。 Na(V)Sp1p 选择性过滤器从 LESWSM 突变为 LDDWSD，这一变化类似于之前显示的改变细菌钠通道 Na(V)Bh1 (NaChBac) 离子选择性的变化，创建了一个钙选择性仅孔通道 Ca(V)Sp1p。我们进一步表明，PDs的产生可以通过从另外两种极端微生物Na(V)s中制备孔蛋白来推广：一种来自碳氢化合物降解剂Alcanivorax borkumensis (Na(V)Ab1p)，另一种来自亚砷酸盐氧化剂Alkalilimnicola ehrlichei (Na(V)Ae1p)。总之，我们的数据建立了一系列活性纯孔离子通道，它们应该是研究控制钠和钙选择性和渗透性的因素的优秀模型系统。此外，我们的研究结果表明，类似的解剖方法可能适用于广泛的 VGIC，因此可以作为简化和加速生物物理、结构和药物开发工作的一种手段。
Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, alpha-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.