| Popis: |
Rab-GTPases exert important roles in secretory vesicle cycles, with Rab3 and Rab27 participating in exocytotic throughput. While Rab3 and Rab27 share many effectors, some are unique, making it critical to identify specific functional roles. Studies on regulation of neurotransmitter release have focused principally on Rab3 and much less on Rab27. Among Rab-GTPases, Rab27 is unique in binding effectors in either the GTP- or GDP-bound state. We here compare secretory responses from ashen mice, which lack functional Rab27a protein, with strain-matched controls. Secretion was evaluated using measurements of membrane capacitance under whole-cell recording in isolated mouse chromaffin cells. UV flash uncaging of Ca2+ raised [Ca2+]i uniformly and induced catecholamine neurotransmitter release. RT-PCR demonstrated the presence of Rab27 in chromaffin cells, and immunoblotting further characterized the expressed isoform as Rab27a, not Rab27b. The exocytic burst (1s post-UV flash) of membrane capacitance increase (ΔCm) was fitted by a double exponential curve, from which the amount and fusion kinetics of granules from the readily- (RRP) and slowly-releasable pools (SRP) were extracted. Exocytic burst ΔCm was greater, and the size of both the RRP and SRP larger, in wild-type cells (RRP, 347 fF; SRP, 616 fF) than in Rab27a-/- cells (RRP, 157fF; SRP, 438 fF), while the kinetics of release from both pools remained unchanged. Expression of Rab27a in Rab27a-/- cells, using electroporation, rescued the wild-type phenotype. Rab 27a is therefore important for priming into the RRP and SRP. To assess dense-core vesicle docking at the plasma membrane, we analyzed granule distribution from E18 chromaffin cell EM micrographs. These data show that while Rab27a-/- cells possess fewer total granules, morphological docking of granules at the plasma membrane is unaffected. Further work is necessary to identify through which effector Rab27a is exerting its effects. |
