Rala Gtpase Tethers Insulin Granules To L- And R-Type Calcium Channels Through Binding 2-1 Subunit

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav) channels. RalA knockdown (KD) in INS-1 cells and primary rat -cells resulted in a reduction in Ca2+ currents arising specifically from L-(Cav1.2 and Cav1.3) and R-type (Cav2.3) Ca2+ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca2+ currents. RalA co-immunoprecipitated with the Cav2-1 auxiliary subunit known to bind the three Cavs. Moreover, the functional molecular interactions between Cav2-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of 2-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and 2-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when 2-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds 2-1 on insulin granules to tether these granules to PM Ca2+ channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.