G protein β subunits regulate Ca v 3.3 T-type channel activity and current kinetics via interaction with the Ca v 3.3 C-terminus

• Published in: Biochimica et biophysica acta. Biomembranes Vol. 1866; no. 6; p. 184337
• Main Authors: Jeong, Sua, Lee, Bo-Young, Rhee, Jeong Seop, Lee, Jung-Ha
• Format: Journal Article
• Language: English
• Published: Netherlands 01.08.2024
• Subjects: Animals; Calcium Channels, R-Type; Calcium Channels, T-Type - chemistry; Calcium Channels, T-Type - genetics; Calcium Channels, T-Type - metabolism; Cation Transport Proteins; GTP-Binding Protein beta Subunits - chemistry; GTP-Binding Protein beta Subunits - genetics; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - chemistry; GTP-Binding Protein gamma Subunits - genetics; GTP-Binding Protein gamma Subunits - metabolism; HEK293 Cells; Humans; Kinetics; Patch-Clamp Techniques; Protein Binding; Rats; G protein βγ subunits; Yeast-two hybrid; Whole cell patch clamp recording; Gating current; Ca(v)3.3 T-type Ca(2+) channel
• ISSN: 1879-2642
• DOI: 10.1016/j.bbamem.2024.184337

Ca influx through Ca 3.3 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 3.3 and the related regulatory pathways remains largely limited. To address this quest, we employed the rat Ca 3.3 C-terminus as bait in yeast-two-hybrid screenings of a cDNA library, identifying rat Gβ as an interaction partner. Subsequent assays revealed that the interaction of Gβ subunit was specific to the Ca 3.3 C-terminus. Through systematic dissection of the C-terminus, we pinpointed a 22 amino acid sequence (amino acids 1789-1810) as the Gβ interaction site. Coexpression studies of rat Ca 3.3 with various Gβγ compositions were conducted in HEK-293 cells. Patch clamp recordings revealed that coexpression of Gβ γ reduced Ca 3.3 current density and accelerated inactivation kinetics. Interestingly, the effects were not unique to Gβ γ but were mimicked by Gβ alone as well as other Gβγ dimers, with similar potencies. Deletion of the Gβ interaction site abolished the effects of Gβ γ . Importantly, these Gβ effects were reproduced in human Ca 3.3. Overall, our findings provide evidence that Gβ(γ) complexes inhibit Ca 3.3 channel activity and accelerate the inactivation kinetics through the Gβ interaction with the Ca 3.3 C-terminus.