Key result
Nav1.5 P627S mutation shifts channel activation depolarizing by ~10 mV, whereas large linker deletions show little effect.
Why the study?
The I-II and II-III cytoplasmic linkers of Nav1.5 are absent from published structures, predicted to be disordered, and their functional role is not well understood.
Do deletions or mutations in the DI-DII linker of the Nav1.5 channel alter its gating function?
Do deletions or mutations in the DI-DII linker of the Nav1.5 channel alter its gating function?
The DI-DII linker of Nav1.5 likely facilitates interactions with other proteins rather than undergoing large conformational shifts, which may aid in evaluating the pathogenicity of variants.
P627S may aid Nav1.5 variant interpretation; hypothesis-generating for linker roles and requires human validation.
The cardiac voltage-gated sodium channel, Nav1.5 initiates the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. The functional Nav1.5 core consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing and a pore domain. The channel also contains three cytoplasmic linkers (I-II, II-III, and III-IV). While Nav1.5 structures have been published, the I-II and II-III linkers have remained absent, are predicted to be disordered, and their functional role is not well understood. We divided the I-II linker into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had unique sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this hypothesis with experiments with individual Nav1.5 constructs with each region deleted. These deletions had small effects on channel gating, though two (430 – 457del and 556 – 607del) reduced peak current. Phylogenetic analysis of the I-II linker revealed five prolines (P627, P628, P637, P640, P648) that were conserved in mammals but absent from the Xenopus sequence. We created mutant channels, where these were replaced with their Xenopus counterparts. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 +/- 2.28 mV). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a significant effect, suggesting that either phosphorylation or another specific serine property is required. Since deletion of large regions had little effect on channel gating while a point mutation had a conspicuous impact, the I-II linker role may be to facilitate interactions with other proteins. Variants may have a larger impact if they create or disrupt these interactions, which may be key in evaluating pathogenicity of variants.
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Wagner et al. (2024) studied Nav1.5 channel function in Xenopus oocytes. Nav1.5 DI-DII linker deletions and point mutations (e.g., P627S) vs. Wild-type Nav1.5 constructs was evaluated on Channel gating and activation (10.13 +/- 2.28 mV). The P627S mutation significantly depolarized Nav1.5 channel activation by 10.13 +/- 2.28 mV, while large linker deletions had little effect.
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