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bioRxiv · 10.1101/2022.06.21.496996

Mapping molecular determinants of Cav2.2 inhibition by RGK proteins and homologs in Xenopus oocytes

Sasson, Y.·Subramaniam, S.·Buki, T.·Almagor, L.·Chomsky-Hecht, O.·Katz, M.·Puhl, H.·Ikeda, S. R.·Dascal, N.·Hirsch, J.
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Abstract

The CaV1 and CaV2 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 1, intracellular {beta} and extracellular 2{delta} subunits, these channels have been targets for pharmacological intervention for decades. Physiological functions of CaV 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 CaV1 and CaV2 function in heterologous expression systems. RGK proteins bind to CaV{beta} and inhibit channel localization and activity by forming a ternary complex with CaV1. Here, we evaluated the influence of RGK proteins on CaV2.2 channels heterologously expressed in Xenopus oocytes. Both Gem and Rad showed no nucleotide dependency on its inhibitory function on CaV2.2. The G-domain and C-terminus could inhibit the CaV2.2 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 CaV{beta}-Gem constructs. We expanded our mapping efforts and prepared various chimeras of Drosophila melanogaster (Dm) RGK sequences fused to CaV{beta} and showed that 22 residues in RGK2t and RGK3L C-terminal imparted complete CaV2.2 inhibition. Point mutations in the DmRGK C-terminus, conserved in mammalian RGK proteins, abrogated the CaV2.2 inhibition to a significant extent, pointing to a hot region in the extreme C-terminus for inhibition of CaV channels. Since RGK homologs are now recognized as physiological modulators in {beta}-adrenergic regulation of CaV channels, the relevance of this curious G-protein family deserves close examination.

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Sasson, Y., Subramaniam, S., Buki, T., Almagor, L., Chomsky-Hecht, O., Katz, M., Puhl, H., Ikeda, S. R., Dascal, N., Hirsch, J.. 2022-06-24. Mapping molecular determinants of Cav2.2 inhibition by RGK proteins and homologs in Xenopus oocytes. https://doi.org/10.1101/2022.06.21.496996

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