Gating properties of specific voltage-gated sodium channel complexes involved in rare disease

About This Grant

ABSTRACT This proposal addresses the need to investigate understudied proteins associated with rare diseases, such as Brugada Syndrome (PAR-25-122). One class of proteins highlighted in this RFA—Scn2b, Scn3b, and Scn4b— belongs to a family of β subunits that associate with the large pore-forming α subunits of voltage-gated sodium channels (NaV), which regulate electrical excitability throughout the body. In total, there are four distinct β subunits that can mix and match with nine different α subunits. Beta subunits are widely recognized for their ability to regulate the gating properties, trafficking, and pharmacology of Nav channel complexes. Dysfunction of these subunits has been linked to several human diseases, including epilepsy and cardiac arrhythmias such as long QT syndrome, atrial fibrillation, and Brugada syndrome. Additionally, mutations in NaV α subunits have been implicated in rare diseases, including SCN8A encephalopathy (SCN8A/ NaV 1.6), hereditary sensory and autonomic neuropathy type 7 (SCN11A/ NaV1.9), and dilated cardiomyopathy-1E (SCN5A/ NaV1.5). A critical step in understanding how beta subunits contribute to disease is elucidating their precise modulatory effects on NaV function. Electrophysiological studies in heterologous cells have demonstrated the ability of beta subunits to influence channel gating, pharmacology, and trafficking. However, results across multiple studies have been inconsistent, often due to variability in the cell lines used. A major confounding factor is that many cell lines endogenously express beta subunits, which can interfere with exogenously introduced β subunits under investigation. To overcome this limitation, we developed a specialized cell line lacking all β subunits, including Scn2b, Scn3b, and Scn4b, as well as Scn1b, MPZ, MPZL1, MPZL2, and MPZL3. These cells, termed beHAPe cells (beta- eliminated haploid cells for expression), provide a controlled system to study NaV channel regulation. Our initial electrophysiological studies using beHAPe cells reveal novel properties of beta subunits in modulating NaV1.5, the primary α subunit in cardiac tissue. Building on these findings, we propose to produce stably-expressing human (HEK) cell lines to systematically define the roles of Scn2b, Scn3b, and Scn4b in modulating additional α subunits, including NaV1.6 (a key subunit in the central nervous system) and NaV1.7, NaV 1.8, and NaV 1.9 (which are predominant in the peripheral nervous system). This work will provide deeper insights into their function in these tissues and their associated diseases. Additionally, our new data suggest that Nav1.8 plays a previously unrecognized role in cardiac function alongside NaV1.5. Understanding how β subunits modulate pore-forming subunits could provide new insights into their involvement in cardiac arrhythmias, expanding their known roles beyond the nervous system. Taken together, in addition to providing new information on the understudied Scn2b, Scn3b, and Scn4b proteins, our newly generated stable cell lines will enable studies for the development of novel therapeutics for isoform specific modulation of specific α- and β-subunit pairs.