RalA GTPase Tethers Insulin Granules to L‐ and R‐Type Calcium Channels Through Binding α2δ‐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 (Ca v ) channels. RalA knockdown (KD) in INS ‐1 cells and primary rat β‐cells resulted in a reduction in Ca 2+ currents arising specifically from L‐(Ca v 1.2 and Ca v 1.3) and R‐type (Ca v 2.3) Ca 2+ channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca 2+ currents. RalA co‐immunoprecipitated with the Ca v α 2 δ‐1 auxiliary subunit known to bind the three Ca v s. Moreover, the functional molecular interactions between Ca v α 2 δ‐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 α 2 δ‐1 to insulin granules without affecting the localization of the other Ca v subunits. Furthermore, we confirmed that RalA and α 2 δ‐1 functionally interact since RalA KD‐induced inhibition of Ca v currents could not be recovered by RalA when α 2 δ‐1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α 2 δ‐1 on insulin granules to tether these granules to PM Ca 2+ 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.