首页> 美国卫生研究院文献>The Journal of Biological Chemistry >A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation
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A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation

机译:毛孔胞质孔处的阻孔疏水基序。 封闭状态Nav1.7通道被 血红蛋白相关的F1449V 突变

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摘要

Sodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7.
机译:钠通道Nav1.7最近引起了人们极大的兴趣,认为它是导致人类疼痛的关键因素。 Nav1.7的功能获得突变产生痛苦的疾病,而Nav1.7的功能丧失突变产生对疼痛的不敏感性。在结构域III /跨膜螺旋S6的C末端内遗传的红血球痛Nav1.7 / F1449V突变使通道激活移动了-7.2 mV,并加速了达到峰值的时间,从而导致伤害感受器过度兴奋。我们基于KcsA钾通道晶体结构构建了Nav1.7的同源性模型,该模型确定了四个系统发育上保守的芳香族残基,它们对应于四个S6螺旋中每个C末端的DIII / F1449。该模型预测,残基1449的侧链大小变化会改变孔的胞质孔径并重塑域间接触表面,从而有助于闭合状态的稳定。为了验证这一假设,我们通过全细胞膜片钳分析比较了野生型和突变Nav1.7通道F1449V / L / Y / W的激活。除F1449V突变以外的所有突变都保留了激活的电压依赖性。与野生型相比,F1449V的峰值时间更短,与 F1449L,但对于F1449Y和F1449W更长,这表明其体积大,疏水性强 残留物对于正常激活是必需的。我们还用 每个结构域中相应的S6芳香残基分别与缬氨酸相连, 模仿自然发生的Nav1.7突变。我们证明 DII / F960V和DIII / F1449V,但不包括DI / Y405V或DIV / F1752V Nav1.7激活,与公认的构象一致 DII和DIII的变化。我们建议四个芳香族残基 到细胞质孔口的门,并且它们的环侧链 形成疏水塞,可稳定 Nav1.7。

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