Cross-species conservation of open-channel block by Na channel β4 peptides reveals structural features required for resurgent Na current.
Cross-species conservation of open-channel block by Na channel β4 peptides reveals structural features required for resurgent Na current.
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DOI：
10.1523/jneurosci.1428-11.2011
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发表时间：
2011-08-10
期刊：
影响因子：
--
通讯作者：
Raman IM
中科院分区：
文献类型：
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作者：
Lewis AH;Raman IM
许多神经元（包括小脑和脑干中的几个）中的电压门控Na通道专门用于允许动作电位的快速放电。重复放电由复苏的Na电流促进，当Na通道从内源性蛋白质的阻断通过开放状态恢复时，Na电流在复极化时流动。迄今为止最好的候选阻断蛋白是NaVβ4。然而，这种蛋白质的序列在物种之间存在差异，同时维持高频放电，提出了NaVβ4胞质尾区的阻断作用是否保守的问题。在这里，我们发现，尽管在NaVβ4序列的差异，从鸡胚分离的浦肯野细胞具有复苏电流的动力学和振幅与小鼠浦肯野细胞中的那些没有区别。此外，来自五个物种的不同NaVβ4胞质尾的合成肽具有在缺乏功能性内源性阻断蛋白的小鼠海马神经元中诱导复活电流的能力。这些数据进一步支持了NaVβ4的阻断作用，也表明了不同残基在诱导开放通道阻断中的相对重要性。为了研究几个高度保守的残基对开放通道阻断的贡献，我们合成了几种突变肽，其中一个苯丙氨酸和两个赖氨酸的身份和相对取向被破坏。这些突变体肽产生的电流与物种衍生的肽相比具有非常不同的动力学，这表明这些残基是开放通道阻断所需的，其近似于生理性复苏的Na电流。因此，如果存在其他阻断蛋白，它们可能与NaVβ4胞质尾区共享这些结构元件。
Voltage-gated Na channels in many neurons, including several in the cerebellum and brainstem, are specialized to allow rapid firing of action potentials. Repetitive firing is facilitated by resurgent Na current, which flows upon repolarization as Na channels recover through open states from block by an endogenous protein. The best candidate blocking protein to date is NaVβ4. The sequence of this protein diverges among species, however, while high-frequency firing is maintained, raising the question of whether the proposed blocking action of the NaVβ4 cytoplasmic tail has been conserved. Here, we find that, despite differences in the NaVβ4 sequence, Purkinje cells isolated from embryonic chick have resurgent currents with kinetics and amplitudes indistinguishable from those in mouse Purkinje cells. Furthermore, synthetic peptides derived from the divergent NaVβ4 cytoplasmic tails from five species have the capacity to induce resurgent current in mouse hippocampal neurons, which lack a functional endogenous blocking protein. These data further support a blocking role for NaVβ4, and also indicate the relative importance of different residues in inducing open-channel block. To investigate the contribution of the few highly conserved residues to open-channel block, we synthesized several mutant peptides in which the identities and relative orientations of a phenylalanine and two lysines were disrupted. These mutant peptides produced currents with vastly different kinetics than did the species-derived peptides, suggesting that these residues are required for an open-channel block that approximates physiological resurgent Na current. Thus, if other blocking proteins exist, they may share these structural elements with the NaVβ4 cytoplasmic tail.