Dose-dependent and Isoform-specific Modulation of Ca²⁺ Channels by RGK GTPases

Although inhibition of voltage-gated calcium channels by RGK GTPases (RGKs) represents an important mode of regulation to control Ca²⁺ influx in excitable cells, their exact mechanism of inhibition remains controversial. This has prevented an understanding of how RGK regulation can be significant in a physiological context. Here we show that RGKs—Gem, Rem, and Rem2—decreased Ca_V1.2 Ca²⁺ current amplitude in a dose-dependent manner. Moreover, Rem2, but not Rem or Gem, produced dose-dependent alterations on gating kinetics, uncovering a new mode by which certain RGKs can precisely modulate Ca²⁺ currents and affect Ca²⁺ influx during action potentials. To explore how RGKs influence gating kinetics, we separated the roles mediated by the Ca²⁺ channel accessory β subunit's interaction with its high affinity binding site in the pore-forming α_1C subunit (AID) from its other putative contact sites by utilizing an α_1C•β3 concatemer in which the AID was mutated to prevent β subunit interaction. This mutant concatemer generated currents with all the hallmarks of β subunit modulation, demonstrating that AID-β–independent interactions are sufficient for β subunit modulation. Using this construct we found that although inhibition of current amplitude was still partially sensitive to RGKs, Rem2 no longer altered gating kinetics, implicating different determinants for this specific mode of Rem2-mediated regulation. Together, these results offer new insights into the molecular mechanism of RGK-mediated Ca²⁺ channel current modulation.