Abstracts

Rationale: Variants in genes encoding G protein βγ subunits (Gβγs) are linked to the developmental and epileptic encephalopathies (DEEs). A current focus of epilepsy research is to understand how variants in non-ion channel genes result in DEE. Previous work has shown that Gβγs can directly modulate the activity of voltage-gated K⁺ and Ca²⁺ channels, however, less well-known is their ability to modulate voltage-gated sodium channel (VGSC) function. VGSCs are responsible for the initiation and propagation of action potentials in neurons. VGSC subtypes are strategically expressed based on their kinetic and voltage-sensing properties to optimize physiological function.

Na_v1.1 and Na_v1.6 are two of the major VGSC a subunits expressed in pediatric brain. The roles of these genes in DEE mechanisms are well known. However, the sensitivity of Na_v1.1 and Na_v1.6 to modulation by Gβγs, as well as how these interactions are regulated by Na_vb1 subunits, is unknown. The goal of our research is to understand the combined mechanisms of Gβγ/Na_vb1 modulation of brain VGSCs. These proposed studies will provide new insights into basic neuromodulatory mechanisms in the brain. They will also uncover novel therapeutic signaling pathways for the treatment of brain disease, including pediatric epilepsy.

Methods: Mass spec protein-profiling
Na_vβ1 was immunoprecipitated in whole brain from postnatal day 18 (P18) Scn1b-V5 epitope tagged mice. Immunoprecipitation samples were analyzed using LC-MS/MS to identify protein binding partners of the Na_vβ1 subunit.

I_Na recordings in HEK cells transiently transfected with Gβγ subunits
Na⁺ current (I_Na) was measured using whole-cell patch clamp. HEK cells stably expressing human Na_v1.1 or Na_v1.6 were transiently transfected with EGFP or Gβ1γ2.

Results: Mass spec protein-profiling
Gγ₂, Gγ₃, Gγ₇ and Gγ₁₂ subunits were found to interact in complex with Na_vβ1 in P18 Scn1b-V5 mouse whole brain.

I_Na recordings in HEK cells transiently transfected with Gβγ subunits
Transient coexpression of Gβ1γ2 in HEK cells stably expressing human Na_v1.6 led to a significant decrease in peak whole cell I_Na density. Coexpression of Gβ1γ2 in HEK cells stably expressing human Na_v1.1 also led to a significant decrease in I_Na density. In contrast to Na_v1.6, the inhibition of Na_v1.1 displayed voltage-dependence, with a significant I_Na density decrease occurring from a prepulse potential of -70 mV, but not -120 mV. No shifts in the voltage-dependence of activation or inactivation of Na_v1.1 or Na_v1.6 were induced by the coexpression of Gβ1γ2 subunits.

Conclusions: Our work shows that Gγ₂, Gγ₃, Gγ₇ and Gγ₁₂ are Na_vb1 binding partners and the neuronal VGSC subtypes Na_v1.1 and Na_v1.6 are relevant effectors of Gβ1γ2 signaling. Transient expression of Gβ1γ2 resulted in significant decreases in Na_v1.1- and Na_v1.6-generated I_Na density in HEK cells. Given the significant role of Gβγs in neurotransmission and neuropharmacology, as well as the roles of Na_v1.1 and Na_v1.6 in controlling neuronal excitability, these data motivate further characterization of this functional interaction in native neuronal preparations.

Funding: Please list any funding that was received in support of this abstract.: Michigan Pharmacology Centennial Fellowship; NIH R37 NS076752.