P/Q Ca2+ Channels are Functionally Coupled to Immediately Releasable Pool Exocytosis in Mouse Chromaffin Cells

YANINA D. ALVAREZ; LORENA I. IBAÑEZ; OSVALDO D. UCHITEL; FERNANDO D. MARENGO

CELL CALCIUM.

Elsevier

Año: 2008 vol. 43 p. 155 - 164

0143-4160

Chromaffin cell exocytosis is triggered by Ca2+ entry through several voltage dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channels types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker ù-conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker ù-Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used á1A subunit knock-out mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3min, 50mM K+) induced similar exocytosis for wild type and knock-out cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knock-out derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells. Keywords: á1A knockout mice; voltage dependent calcium channels; stimulus-secretion coupling.