“Synaptic protein interaction site modulates P/Q Ca2+ current and exocytosis of Immediately Releasable Pool in mouse chromaffin cells

YANINA D. ALVAREZ; A. E .PEREZ BAY; SCOTT E.JAVIS; H. W.TEDFORD; GERALD ZAMPONI; FERNANDO D. MARENGO

Mexico

Simposio; 15th International Symposium on Chromaffin Cell Biology; 2009

&amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;!-- /* Font Definitions */ @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-1610611985 1107304683 0 0 159 0;} @font-face {font-family:Geneva; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-alt:Arial; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-format:other; mso-font-pitch:variable; mso-font-signature:3 0 0 0 1 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:6.0pt; margin-left:0cm; text-align:justify; text-indent:35.45pt; mso-pagination:widow-orphan; font-size:12.0pt; mso-bidi-font-size:10.0pt; font-family:"Geneva","sans-serif"; mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:EN-US; mso-fareast-language:EN-US;} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --&amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 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.