G protein β subunits regulate Cav3.3 T-type channel activity and current kinetics via interaction with the Cav3.3 C-terminus

Ca²⁺ influx through Ca_v3.3 T-type channel plays crucial roles in neuronal excitability and is subject to regulation by various signaling molecules. However, our understanding of the partners of Ca_v3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Ca_v3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gβ₂ as an interaction partner. Subsequent assays revealed that the interaction of Gβ₂ subunit was specific to the Ca_v3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789–1810) as the Gβ₂ interaction site. Coexpression studies of rat Ca_v3.3 with various Gβγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gβ₂γ₂ reduced Ca_v3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gβ₂γ_2, but were mimicked by Gβ₂ alone as well as other Gβγ dimers, with similar potencies. Deletion of the Gβ₂ interaction site abolished the effects of Gβ₂γ₂. Importantly, these Gβ₂ effects were reproduced in human Ca_v3.3. Overall, our findings provide evidence that Gβ(γ) complexes inhibit Ca_v3.3 channel activity and accelerate the inactivation kinetics through the Gβ interaction with the Ca_v3.3 C-terminus.