Voltage-gated sodium channel (VGSC) beta (β) subunits have been called the "overachieving" auxiliary ion channel subunit. Indeed, these subunits regulate the trafficking of the sodium channel complex at the plasma membrane and simultaneously tune the voltage-dependent properties of the pore-forming alpha-subunit. It is now known that VGSC β-subunits are capable of similar modulation of multiple isoforms of related voltage-gated potassium channels, suggesting that their abilities extend into the broader voltage-gated channels. The gene family for these single transmembrane immunoglobulin beta-fold proteins extends well beyond the traditional VGSC β1-β4 subunit designation, with deep roots into the cell adhesion protein family and myelin-related proteins - where inherited mutations result in a myriad of electrical signaling disorders. Yet, very little is known about how VGSC β-subunits support protein trafficking pathways, the basis for their modulation of voltage-dependent gating, and, ultimately, their role in shaping neuronal excitability. An evolutionary approach can be useful in yielding new clues to such functions as it provides an unbiased assessment of protein residues, folds, and functions. An approach is described here which indicates the greater emergence of the modern β-subunits roughly 400 million years ago in the early neurons of Bilateria and bony fish, and the unexpected presence of distant homologues in bacteriophages. Recent structural breakthroughs containing α and β eukaryotic sodium channels containing subunits suggest a novel role for a highly conserved polar contact that occurs within the transmembrane segments. Overall, a mixture of approaches will ultimately advance our understanding of the mechanism for β-subunit interactions with voltage-sensor containing ion channels and membrane proteins.

Voltage-gated, sodium ion-selective channels (Na V ) generate electrical signals contributing to the upstroke of the action potential in animals. Na V s are also found in bacteria and are members of a larger family of tetrameric voltagegated channels that includes Ca V s, K V s, and Na V s. Prokaryotic Na V s likely emerged from a homotetrameric Ca 2+ -selective voltage-gated progenerator, and later developed Na + selectivity independently. The Na V signaling complex in eukaryotes contains auxiliary proteins, termed beta (β) subunits, which are potent modulators of the expression profiles and voltage-gated properties of the Na V pore, but it is unknown whether they can functionally interact with prokaryotic Na V channels. Herein, we report that the eukaryotic Na V β1-subunit isoform interacts with and enhances the surface expression as well as the voltage-dependent gating properties of the bacterial Na V , NaChBac in Xenopus oocytes. A phylogenetic analysis of the β-subunit gene family proteins confirms that these proteins appeared roughly 420 million years ago and that they have no clear homologues in bacterial phyla. However, a comparison between eukaryotic and bacterial Na V structures highlighted the presence of a conserved fold, which could support interactions with the β-subunit. Our the electrophysiological, biochemical, structural and bioinformatics results suggests that the prerequisites for β-subunit regulation are an evolutionarily stable and intrinsic property of some voltage-gated channels.

Voltage-gated sodium channels control the upstroke of the action potential in the excitable cells of nerve and muscle. Inherited mutations in these proteins are a primary cause of epilepsy, chronic pain and cardiac arrhythmia, thus making sodium channels major therapeutic targets. The sodium channel signaling complex in animals is comprised of a single sodium ion conducting, pore-forming transmembrane protein and multiple auxiliary subunits. These auxiliary subunits are known to alter the function and expression profiles of the pore-forming subunit. The biological roles for auxiliary subunits on channel function and expression of the pore subunit are poorly understand, as is the composition and stoichiometry of auxiliary subunits within the channel complex. The focus of my thesis is to generate biochemical assays and to use these approaches to advance the understanding of the interactions between different types of voltage-gated channels and their auxiliary subunits. To this end, a novel interaction was identified between the eukaryotic sodium channel auxiliary subunits and a prokaryotic voltage-gated sodium channel, a protein that diverged from the eukaryotic voltage-gated sodium channels billions of years ago. The interactions between the auxiliary subunits and channels were probed extensively with chemical and photochemical crosslinkers in search of interaction surfaces and to provide plausible mechanisms of interaction. From this work, and through observations in the published literature, a rudimentary concept emerges that the voltage-gated sodium channel auxiliary subunit gene family may serve multiple roles in affecting electrical signaling by associating with different types of voltage-gated ion channels.