Alternative Splicing in the Voltage-Gated Sodium Channel DmNa_vRegulates Activation, Inactivation, and Persistent Current

Abstract

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 (DmNav, 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 (V1/2), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V1/2 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.

Drosophila as a Model for Epilepsy:bssIs a Gain-of-Function Mutation in the Para Sodium Channel Gene That Leads to Seizures

“…Constructs utilized the cDNA of the Na V 1-1 para isoform, the most common splice variant in adults (Olson et al 2008;Lin et al 2009), expressed in all neurons with an Elav-Gal4 c155 driver. Bang-sensitive paralysis was completely suppressed (0% BS) in para bss1 /1 heterozygotes carrying two copies of the transgene; in comparison, sibling controls showed 78.3% BS ( Figure 5A) (test genotype: Elav-Gal4 C155 para bss1 /1; UAS-Na V 1-1 x2; control genotype: Elav-Gal4 C155 para bss1 /1).…”

Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

“…If the inactivation process is hampered, various physiological problems appear, like cardiac arrest, hyper excitability, hysteria etc, due to the persistent current. 5,6,7,8 In particular, sodium channels are targeted for anesthesia and treatments for genetic diseases in the brain, skeletal muscles, and heart. 9 The physiological importance of the voltage gated sodium channel is associated with numerous pathologies namely cardiovascular, neuronal, neuromuscular, musculoskeletal, metabolic, and respiratory systems 10,11 due to the inherited ion channel diseases due to mutations.…”

Preparation of <em>Drosophila</em> Central Neurons for <em>in situ</em> Patch Clamping

“…Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain 3,4 and ventral nerve cord of embryonic 5,6 , larval 7,8,9,10 , and adult Drosophila 11,12,13,14 . A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents.…”