Synaptic protein interaction site modulates P/Q Ca2+ current and exocytosis of immediately releasable pool in mouse chromaffin cells.

YANINA D. ALVAREZ; A. PEREZ BAY; S.E. JAVIS; H.W. TEDFORD; G. ZAMPONI; F.D. MARENGO

Mérida, México.

Congreso; 15th Internacional Symposium of Chromaffin Cell Biology.; 2009

Internacional Symposium of Chromaffin Cell Biology

SYNAPTIC PROTEIN INTERACTION SITE MODULATES P/Q Ca2+ CURRENT AND EXOCYTOSIS OF IMMEDIATELY RELEASABLE POOL IN MOUSE CHROMAFFIN CELLS. Yanina D. Álvarez1, Andrés Perez Bay1, Scott E.Javis2, H. W.Tedford2, Gerald Zamponi2 and Fernando D. Marengo1 1Laboratorio de Fisiología y Biología Molecular, Facultad de Ciencias Exactas y Naturales, IFIByNE (UBA-CONICET). Buenos Aires, Argentina. 2University of Calgary. School of Medicine. Calgary, Canada. Immediately Releasable Pool (IRP) is a small group of ready releasable vesicles that can be released by short pulses because of its proximity to calcium channels. In a previous work we used specific pharmacological Ca2+ channels blockers and α1A subunit KO mice to demonstrate that IRP exocytosis is specifically coupled to P/Q Ca2+ current [1]. This coupling raises the question of how the interaction between IRP vesicles and P/Q Ca2+ channels is produced. The synprint sequence located in the intracellular loop between II and III region of the α1 subunit of  P/Q and N Ca2+ channels can interact with proteins of the exocytic machinery, regulating channels localization, modulating the Ca2+ currents, and being determinant in vesicle-channel coupling associated to fast exocytosis [2]. We wonder if synprint sequence is responsible for the tight coupling between IRP exocytosis and P/Q Ca2+ current in mice chromaffin cells. IRP was estimated by upper (Bmax) and lower (Bmin) bounds [3]. We first confirmed that, as it is expected, IRP was significantly reduced in the presence of a  fast Ca2+ buffer BAPTA in comparison to the slow buffer EGTA (p<0.05) and that the specific P/Q channel blocker ω-agatoxin-IVA (200 nM) almost abolished IRP exocytosis (17 % respect to control conditions). Next, we transfected chromaffin mouse cells with IRES plasmids containing the synprint peptide sequence (to interfere with channel-exocytic proteins interaction) and EGFP. Synprint transfected cells (syn cells) showed a tendency to have smaller Ca2+ current (ICa2+) densities than control cells and presented a clear reduction on IRP exocytosis (to 40% of control values; p<0.005). The application of the specific P/Q blocker ω-agatoxin IVA on syn cells produced no additional changes neither on ICa2+ nor IRP exocytosis. The ICa2+ versus applied voltage curve (50 ms depolarization) of syn cells was reduced in comparison with control cells (p < 0.02, at 0 mV) to a similar level than ω-agatoxin IVA treated control cells. On the other hand, the addition of the L channel blocker nitrendipine (10 mM) on syn cells certainly provoked an additional reduction of ICa2+ (p<0.001). In consequence, our data show that the exogenous synprint is reducing P/Q calcium current, and in consequence supressing IRP exocytosis, in agreement with our previous results. A possible mechanism  would be the interference of exogenous synprint in the traffic of P/Q channels to plasmamembrane. 1. Álvarez YD et al, 2008. 2. Davies JN and Zamponi G W, 2008. 3. Voets T  et al, 1999.