Mapping molecular determinants of Ca _V 2.2 inhibition by RGK proteins and homologs in Xenopus oocytes

The Ca _V 1 and Ca _V 2 families of voltage-dependent calcium channels play a crucial role in neurotransmitter release, excitation-contraction and many other cellular processes. Comprised of the membrane pore-forming α ₁ , intracellular β and extracellular α ₂ δ subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of Ca _V channels are attenuated by either constitutively or transiently bounds proteins in the cellular environment. The RGK (Rad, Gem, Rem, and Rem2) G-protein family potently inhibits Ca _V 1 and Ca _V 2 function in heterologous expression systems. RGK proteins bind to Ca _V β and inhibit channel localization and activity by forming a ternary complex with Ca _V α ₁ . Here, we evaluated the influence of RGK proteins on Ca _V 2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on Ca _V 2.2. The G-domain and C-terminus could inhibit the Ca _V 2.2 channel independently when co-expressed with channel subunits. Our results demonstrated that structural determinants in Gem, crucial for channel inhibition, lie within the 222-296 amino acid region containing both the partial G-domain and C-terminus as determined from chimeric Ca _V β-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster ( Dm ) RGK sequences fused to Ca _V β and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete Ca _V 2.2 inhibition. Point mutations in the Dm RGK C-terminus, conserved in mammalian RGK proteins, abrogated the Ca _V 2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of Ca _V channels. Since RGK homologs are now recognized as physiological modulators in β-adrenergic regulation of Ca _V channels, the relevance of this curious G-protein family deserves close examination.