Fletcher, E.V.; (2011) Alternative splicing in SCN1A: biophysical consequences for NaV1.1 channels. Doctoral thesis , UCL (University College London).

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Abstract

NaV1.1 is a voltage-gated sodium channel encoded by the gene SCN1A. Mutations in SCN1A cause dominantly inherited epilepsy syndromes in humans and NaV1.1 is an important target of several anti-epileptic drugs (AEDs). A common polymorphism in this gene has been shown to alter the expression of two splice variants of the channel, NaV1.1-5N (containing exon 5N) and NaV1.1-5A (containing exon 5A). Although the splicing is highly conserved and the polymorphism that modifies it has been associated with altered AED dosage, the functional impact of the splicing on NaV1.1 is unknown. This project used whole cell voltage clamp of heterologously-expressed NaV1.1-5A and 5N to compare the intrinsic properties of the splice variants, their modulation by AEDs, their interaction with a published epilepsy mutation (R1648H), their modulation by G-proteins and how they responded to co-expression of sodium channel β subunits. The main finding was that, although when recorded at physiological temperatures the splice variants produced macroscopic currents that were similar for many parameters, they differed in the rate at which they recovered from inactivation, with NaV1.1-5N recovering more rapidly than NaV1.1-5A. This difference in recovery was conferred by a single amino acid substitution that is conserved in several sodium channels that are alternatively spliced at this site, and the difference was obscured in the presence of the common AED, phenytoin. Inclusion of the mutation R1648H also eradicated the difference by disproportionately slowing the recovery of NaV1.1-5N. Although several other subtle differences were seen, no consistent differences were found in interactions between the splice variants and G-proteins or β subunits. By converging on a single parameter, recovery from inactivation, the data presented here suggest this parameter is modulated by splicing, and may play a role in development and treatment of seizures.

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