Role of Rab11a in the regulated secretory pathway in chromaffin cells

ALFONSO BUENO, SAMUEL A.; MARENGO, FERNANDO D.; GALLO, LUCIANA I.

CABA

Congreso; SAN2022 Meeting; 2022

Sociedad Argentina de investigación en neurociencias

The small GTPase Rab11a is a well-known coordinator of constitutive recycling pathways. Little is known about its role in the secretory pathways. Therefore, to fill this gap, we studied the Rab11a’s role in the regulated secretion using murine chromaffin cells, a well-known cellular system for neurosecretion. We transfected fluorescently-tagged Rab11a wild-type (WT), constitutively active Rab11aQ70L mutant (Q70L) or dominant negative Rab11aS25N mutant (S25N). Regulated secretion was studied by electrophysiology, registering real-time capacitance changes with the patch-clamp technique in whole-cell configuration. Upon individual or train depolarization stimuli, Rab11a mutants reduced the exocytosis, while calcium currents were not significantly affected. To further confirm these results, independently of an effect on calcium channels, we evaluated capacitance changes after dialyzing 1.5 µM free calcium. In these conditions, maximum velocity of exocytosis was reduced for both mutants. 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 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 in chromaffin cells.