Kinetics of Rab27a-dependent actions on vesicle docking and priming in pancreatic beta-cells

The small GTPase Rab27a, along with the isoforms of Rab3, is present on insulin secretory granules and has been implicated in regulation of Ca(2+)-triggered exocytosis. We have used membrane capacitance measurements to define the role of Rab27a in regulating the size and refilling of distinct pools of insulin granules by comparison of evoked secretory responses from Rab27a-null ashen and strain-matched wild-type control pancreatic beta-cells. We find that ashen beta-cells display a kinetic defect in refilling of readily releasable and immediately releasable vesicle pools (RRP and IRP, respectively) in response to depolarization-evoked Ca(2+) influx. The deficit in IRP refilling was not observed in the presence of stimulatory glucose concentrations (16.7 mm), though incomplete refilling of the RRP persisted. Comparatively, beta-cells from Rab3a(-/-) mice exhibited complete refilling of the IRP and RRP, demonstrating that Rab27a and Rab3a exert distinct roles in the insulin granule secretory pathway. Further, depletion of the RRP in ashen beta-cells was twofold faster than that of control beta-cells. These deficits in refilling and exocytotic rate in ashen beta-cells were absent when cAMP-regulatory pathways were activated. Elevated cAMP increased the RRP pool size, and complete refilling of the RRP occurred in ashen beta-cells; responses were comparable to wild-type controls. These effects of cAMP were largely eliminated by Rp-cAMP inhibition of PKA, indicating that PKA acts on vesicle priming downstream or via pathways independent of Rab27a. In summary, Rab27a exerts dual roles in glucose-mediated insulin granule exocytosis, facilitating refilling of releasable granule pools while also limiting the rate of release from these pools.