Alternative Splicing in the Voltage-Gated Sodium Channel DmNav Regulates Activation, Inactivation, and Persistent Current
Alternative Splicing in the Voltage-Gated Sodium Channel DmNav Regulates Activation, Inactivation, and Persistent Current
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DOI：
10.1152/jn.00613.2009
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发表时间：
2009-09-01
影响因子：
2.5
通讯作者：
Baines, Richard A.
中科院分区：
文献类型：
--
作者：
Lin, Wei-Hsiang;Wright, Duncan E.;Baines, Richard A.
Lin W-H，Wright DE，Muraro NI，Baines RA.电压门控钠通道DmNa（v）中的选择性剪接调节激活、失活和持续电流。神经生理学杂志102：1994-2006，2009。首次发表于2009年7月22日; doi：10.1152/jn.00613.2009。神经元信号的多样性不仅是差异基因表达的产物，也是选择性剪接的产物。然而，虽然公认的，在离子通道转录选择性剪接通道动力学的确切贡献仍然知之甚少。无脊椎动物的基因组较小，为研究剪接对神经元功能的贡献提供了有吸引力的模型。在这项研究中，我们报告的唯一的电压门控Na+基因（DmNa（V），麻痹）的选择性剪接变体的测序和生物物理特性，在后期胚胎的果蝇。我们确定了27个独特的剪接变异，基于15个替代外显子的存在。在非洲爪蟾卵母细胞中的异源表达表明，替代外显子j、e和f主要影响激活动力学：当存在时，外显子f赋予半激活电压（V-1/2）的超极化移位，而j和e导致去极化移位。外显子h的存在足以在稳态失活的V-1/2中产生去极化移位。持续性Na+电流的大小，但不是快速失活电流，在体内的卵母细胞和果蝇运动神经元的直接影响的存在下，一对相互排斥的，跨膜外显子，称为k和L。与含有L的转录本相比，含有k的转录本具有显著较小的持续电流。最后，我们表明，缺乏所有细胞质选择性剪接外显子的转录物仍然产生功能通道，表明剪接不仅可以通过改变蛋白质结构来影响通道动力学，还可以通过允许差异修饰（即，磷酸化、辅因子的结合等）。我们的研究结果提供了一个功能的基础，了解选择性剪接的电压门控Na+通道的结果在神经元信号的多样性。
Lin W-H, Wright DE, Muraro NI, Baines RA. Alternative splicing in the voltage-gated sodium channel DmNa(v) regulates activation, inactivation, and persistent current. J Neurophysiol 102: 1994-2006, 2009. First published July 22, 2009; doi:10.1152/jn.00613.2009. Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. In this study we report the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na+ gene (DmNa(v), paralytic), in late-stage embryos of Drosophila melanogaster. We identify 27 unique splice variants, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j, e, and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage (V-1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V-1/2 of steady-state inactivation. The magnitude of the persistent Na+ current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L. Transcripts containing k have significantly smaller persistent currents compared with those containing L. Finally, we show that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). Our results provide a functional basis for understanding how alternative splicing of a voltage-gated Na+ channel results in diversity in neuronal signaling.