Cellular and Molecular Neurobiology
Author: Samuel Alberto Alfonso Bueno | email: alfonsosamuel25@gmail.com
Samuel A. Alfonso-Bueno 1°, Fernando D. Marengo 1°, Luciana I. Gallo 1°
1° Instituto de Fisiología, Biología Molecular y Neurociencias (UBA-CONICET). Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires
The small GTPase Rab11a is a well-known coordinator of constitutive recycling pathways. Little is known about its role in secretory pathways. To fill this gap, we studied the Rab11as role in the regulated neurosecretion in murine chromaffin cells. We transfected fluorescently-tagged Rab11a wild-type (WT), constitutively active Rab11aQ70L mutant (Q70L) or dominant negative Rab11aS25N mutant (S25N). Regulated exocytosis was registered as real-time capacitance changes with the patch-clamp technique in whole-cell configuration. Upon individual or short train depolarization stimuli, all Rab11a variants reduced the exocytosis respect to control, but no differences were obtained between mutants and WT. To study the effect of both mutants on massive exocytosis, we evaluated capacitance changes after dialyzing cells with 1.5 µM free calcium. In these conditions, maximum velocity (fF/s) of exocytosis was reduced for S25N (19±2) and Q70L (19±2) when compared with WT (32±4) or control cells (43±2). To evaluate changes on the number of secretory vesicles, we cotransfected either Rab11a variant (WT, S25N and Q70L) with fluorescently-tagged neuropeptide Y (NPY), as a marker for regulated secretory vesicles. By confocal microscopy, we observed a significant reduction in the amount of peripheral NPY in cells expressing S25N, an effect that was stronger after high K+ stimulation. We conclude that Rab11a regulates secretory exocytosis by modulating the availability of secretory vesicles.