Rapid endocitosis in mouse chromaffin cells

A. VER¨®NICA BELINGHERI; FERNANDO D. MARENGO

Huerta Grande. C¨®rdoba.

Congreso; 1ra Reuni¨®n Conjunta de la Sociedad Argentina de Neurociencias y Taller de Neurociencias; 2009

Sociedad Argentina de Neurociencias y Taller Argentino de Neurociencias

Rapid Endocytosis in Mouse Chromaffin Cells Ana Ver¨®nica Belingheri, Fernando Marengo. Laboratorio de Fisiolog¨ªa y Biolog¨ªa Molecular (LFBM), Instituto de Fisiolog¨ªa, Biolog¨ªa Molecular y Neurociencia (IFIByNE), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. CONICET   Endocytosis is critical for maintaining membrane homeostasis and secretion reliability in neuroendocrine cells. We studied rapid endocytosis in mouse chromaffin cells using patch-clamp - whole-cell capacitance measurements. Single or repetitive depolarizations were applied, but the process was consistently activated only with unique pulses lasting ¡Ý0.5s. Two patterns were identified: (1) the amount of membrane retrieved was the same than the one added by exocytosis (compensatory endocytosis, CE), or (2) membrane retrieval was bigger than endocytosis (excess retrieval, ER). CE occurred at relatively low Ca2+ entry, but ER was triggered by higher Ca2+ influx (45.3¡À9.2pC vs 114.23¡À15.3pC, p<0.005).  To evaluate if the measured endocytosis is carried out by more than one component, multiexponential fittings were performed. A frequency histogram showed three time constants populations: two components associated to rapid endocytosis, tR1<2.5s and tR2 between 2.5 and 16s, and one to slow endocytosis tL >16s. ER showed a faster tR1 component (0.40¡À0.06 vs 1.20¡À0.43s, p<0.01) and bigger contribution of two rapid endocytosis components to the whole process (21.9¡À5.1 vs 6.7¡À4.7%, p<0.05; 68.6¡À6.7 vs 41.8¡À9.9%, p<0.05) in comparison with CE. Our data suggest that endocytosis is a complex phenomenon, in which ER is favored by a high Ca2+ entry and is driven by both fast components. Finally, in order to study the fate of the internalized membrane we performed experiments with FM1-43. We found that after high K+ depolarizations inducing excess retrieval (Endo-Exo) of 12.2¡À4.0% (capacitance gave a similar value: 10¡À2.5%), approximately the 50% of endocytosed membrane was recycled to releasable vesicles.