Abstract:
The sodium (Na+) leak channel (NALCN) is a member of the four-domain voltage-gated cation channel family that includes the prototypical voltage-gated sodium and calcium channels (NaVs and CaVs, respectively). Unlike NaVs and CaVs, which have four lateral fenestrations that serve as routes for lipophilic compounds to enter the central cavity to modulate channel function, NALCN has bulky residues (W311, L588, M1145, and Y1436) that block these openings. Structural data suggest that occluded fenestrations underlie the pharmacological resistance of NALCN, but functional evidence is lacking. To test this hypothesis, we unplugged the fenestrations of NALCN by substituting the four aforementioned residues with alanine (AAAA) and compared the effects of NaV, CaV, and NALCN blockers on both wild-type (WT) and AAAA channels. Most compounds behaved in a similar manner on both channels, but phenytoin and 2-aminoethoxydiphenyl borate (2-APB) elicited additional, distinct responses on AAAA channels. Further experiments using single alanine mutants revealed that phenytoin and 2-APB enter the inner cavity through distinct fenestrations, implying structural specificity to their modes of access. Using a combination of computational and functional approaches, we identified amino acid residues critical for 2-APB activity, supporting the existence of drug binding site(s) within the pore region. Intrigued by the activity of 2-APB and its analogues, we tested compounds containing the diphenylmethane/amine moiety on WT channels. We identified clinically used drugs that exhibited diverse activity, thus expanding the pharmacological toolbox for NALCN. While the low potencies of active compounds reiterate the pharmacological resistance of NALCN, our findings lay the foundation for rational drug design to develop NALCN modulators with refined properties.
摘要:
钠(Na)泄漏通道(NALCN)是四域电压门控阳离子通道家族的成员,其中包括典型的电压门控钠和钙通道(NaVs和CaVs,分别)。与Navs和Cav不同,它们有四个侧开窗,作为亲脂性化合物进入中央腔调节通道功能的途径,NALCN具有阻塞这些开口的大体积残基(W311、L588、M1145和Y1436)。结构数据表明,封闭的开窗是NALCN的药理学抗性的基础,但缺乏功能性证据.为了检验这个假设,我们通过用丙氨酸(AAAA)代替上述四个残基来消除NALCN的开窗,并比较了NaV的作用,CaV,野生型(WT)和AAAA通道上的NALCN阻断剂。大多数化合物在两个通道上的行为方式相似,但苯妥英和2-氨基乙氧基二苯基硼酸酯(2-APB)引起了额外的,AAAA频道上的不同响应。使用单一丙氨酸突变体的进一步实验表明,苯妥英和2-APB通过不同的开窗进入内腔,暗示他们的访问模式的结构特异性。使用计算和函数方法的组合,我们确定了对2-APB活性至关重要的氨基酸残基,支持孔区域内药物结合位点的存在。对2-APB及其类似物的活性感兴趣,我们在WT通道上测试了含有二苯基甲烷/胺部分的化合物。我们确定了临床使用的药物,表现出不同的活性,从而扩大NALCN的药理学工具箱。虽然活性化合物的低效力重申了NALCN的药理抗性,我们的研究结果为合理设计药物以开发具有精细特性的NALCN调节剂奠定了基础.