Molecular Determinants of Gem Protein Inhibition of P/Q-type Ca2+ Channels

The RGK family of monomeric GTP-binding proteins potently inhibits high voltage-activated Ca2+ channels. The molecular mechanisms of this inhibition are largely unclear. In Xenopus oocytes, Gem suppresses the activity of P/Q-type Ca2+ channels on the plasma membrane. This is presumed to occur through direct interactions of one or more Gem inhibitory sites and the pore-forming Cav2.1 subunit in a manner dependent on the Ca2+ channel subunit β (Cavβ). In this study we investigated the molecular determinants in Gem that are critical for this inhibition. Like other RGK proteins, Gem contains a conserved Ras-like core and extended N and C termini. A 12-amino acid fragment in the C terminus was found to be crucial for and sufficient to produce Cavβ-dependent inhibition, suggesting that this region forms an inhibitory site. A three-amino acid motif in the core was also found to be critical, possibly forming another inhibitory site. Mutating either site individually did not hamper Gem inhibition, but mutating both sites together completely abolished Gem inhibition without affecting Gem protein expression level or disrupting Gem interaction with Cav2.1 or Cavβ. Mutating Gem residues that are crucial for interactions with previously demonstrated RGK modulators such as calmodulin, 14-3-3, and phosphatidylinositol lipids did not significantly affect Gem inhibition. These results suggest that Gem contains two candidate inhibitory sites, each capable of producing full inhibition of P/Q-type Ca2+ channels. Background: Gem, an RGK protein, strongly inhibits P/Q-type Ca2+ channels. Results: A 12-amino acid C-terminal fragment and a 3-amino acid motif in the core of Gem play an important role in this inhibition. Conclusion: Gem possesses two candidate inhibitory sites. Significance: This work identifies key molecular determinants of Ca2+ channel inhibition by RGK proteins, offering opportunities to modulate this inhibition.