Exocytosis of Immediately Releasable Pool is Coupled with P/Q Ca2+ Through a Specific Interaction Mechanism.

YANINA D. ÁLVAREZ; A. PEREZ BAY; L.I. IBÁÑEZ; G. ZAMPONI; F.D. MARENGO

Buzios, Río de Janeiro, Brasil

Congreso; I Congreso IBRO/LARC de Neurociencias de América Latina, Caribe y Península Ibérica.; 2008

IBRO/LARC

Chromaffin cell exocytosis is triggered by a localized intracellular Ca2+ increase due to the activation of voltage dependent calcium channels. It was described that a small population of the ready releasable pool (RRP) called immediately releasable pool (IRP), can be released by short pulses, supposedly because a close proximity to calcium channels. We used capacitance measurements on cultured chromaffin mouse cell, and IRP was estimated to be 31±3fF for control conditions, what represents the 25% of RRP. Channels blockers and P/Q KO mice’s let us to demonstrate that P/Q are the dominant calcium channels associated to the release of IRP in mouse chromaffin cells. A possible hypothesis is that the coupling between P/Q channel and the IRP vesicles could be dependent of the synaptic protein interaction site, synprint, present in á1 subunit. This site interacts with key vesicular and exoctytotic proteins. Chromaffin cells were transfected with pIRES-EGFP with inserted synprint cDNA. We expected that free exogenous synprint peptide will compete with the endogenous synprint of P/Q channels, loosing the coupling between IRP vesicles and those channels. Expression of synprint peptide induced a significative reduction in the IRP exocytosis (31±4fF vs 10±4fF, p<0.05). As expected, application of P/Q blocker wagatoxin-IVA on transfected cells did not induced additional reduction of IRP.  Stronger stimulation protocols designed to release vesicles form whole RRP on transfected cells did not show a significative effect. This results support the idea that the IRP vesicles are physically coupled to the P/Q channels.  Different distributions of channels and RRP vesicles are modeled in order to reproduce the proportion of vesicles in close proximity to the channels that yield the presence of IRP:  A) a uniform and random distribution channels and vesicles, B) a fix grid for the calcium channels and random vesicles distribution, and C) nonuniform distributions in which a fix percentage of RRP vesicles are forced to be placed at no more than 30nm distance from channels. In the first two conditions only 6% of RRP vesicles were associated to IRP. On the other hand, the model supports that a IRP of approximately 25% RRP can be explained by some type of specific interaction restriction (C).