Date of Award
12-23-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
The cardiac voltage-gated sodium channel, Nav1.5 is essential for initiating the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. Like many channels, its known properties depend heavily on its structure. The functional core of the alpha subunit consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing domain and a pore domain. The channel also contains two cytoplasmic termini and three cytoplasmic linkers (I-II, II-III, and III-IV). While most of the channel has been resolved in multiple structures, the I-II and II-III linkers have remained conspicuously absent and are predicted to be disordered. Though some information regarding their roles in regulation and various interactions is available, the definitive role of these linkers is not well understood. We investigated the basic functional role of the I-II linker by starting with a detailed analysis of its sequence. We separated it into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had such distinct sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this with experiments where we created individual Nav1.5 constructs with each region deleted, expressed them in Xenopus oocytes, and recorded the current with a COVG voltage-clamp. Interestingly, these deletions had very little effect on channel gating, though at least two (430 – 457del and 556 – 607del) resulted in a significant reduction in peak current. Disordered proteins and regions are typically less conserved overall than ordered proteins in terms of primary sequence conservation. In fact, there is some evidence that molecular features, such as residue content and net charge, may be more important for functional conservation than the exact sequence. We performed a multi-species sequence analysis and found that the different regions of the I-II linker had varying degrees of conservation. We also found that certain regions had noticeably more varied proline fractions that were higher on average for mammals than for non-mammals. Further, we identified charged residues and several interaction sites as exhibiting high levels of conservation. The pathogenicity of variants within the I-II linker is difficult to evaluate, so we attempted to correlate in-silico conformational changes for channel variants to in-vitro changes in channel function. We used a Metropolis Monte Carlo scheme to simulate portions of the I-II linker containing the variants. First, we investigated four clusters of phosphorylation sites in the I-II linker: S457-460, S483-486, S497-499, and S664-671. Comparing simulations of the wild-type sequence with simulations of an increasing number of phosphomimetic mutations, we found that only segments containing the S497-499 and S664-671 clusters showed conformational sensitivity to the mutations, while those containing S457-460 and S483-486 were unaffected. In Lorenzini et al. 2021, it was found that only mutations to S664-671 affected channel function when expressed in HEK293 cells. This experimental data is consistent with the simulations for S457-460, S483-486, and S664-671 but not for the S497-499. Additionally, we investigated five prolines (P627, P628, P637, P640, P648) that had been revealed in a multi-species sequence analysis to be conserved in mammals but notably absent from the Xenopus sequence. Simulations suggested that the presence of these prolines could have an expansionary effect on the conformational space occupied by the protein. We created mutant channels, where we replaced all or some of these prolines with their Xenopus counterparts. Ionic currents from mutant channels were recorded with the COVG voltage-clamp in Xenopus laevis oocytes. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 +/- 2.28 mV depolarizing activation V50 shift). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a statistically significant effect, suggesting that either phosphorylation or another specific property of serine may be required to observe the effect. Since the deletion of large regions from the linker had little effect on channel gating while single interaction-associated mutations had conspicuous impacts, the I-II linker’s fundamental role may be to act as an interface for interactions with other proteins. If this is the case, without the specific interacting proteins present, removing the regions would likely have little effect on channel function, similar to what we observed in our deletion recordings in oocytes. Variants may have a larger impact if they create or disrupt these interactions. Determining whether they do so may be key in evaluating the likely pathogenicity of variants.
Language
English (en)
Chair
Jonathan Silva
Committee Members
Alex Holehouse; Colin Nichols; Janice Robertson; Michael Vahey