It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca2+ channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca2+ current. Accordingly, in the present work we found that the Ca2+ current flowing through P/Q-type Ca2+ channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca2+ current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K+ stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca2+ channels.

Perez Bay AE, Ibañ ez LI, Marengo FD. Rapid recovery of releasable vesicles and formation of nonreleasable endosomes follow intense exocytosis in chromaffin cells. Am J Physiol Cell Physiol 293: C1509-C1522, 2007. First published August 8, 2007 doi:10.1152/ajpcell.00632.2006.-Neurons and neuroendocrine cells must retrieve plasma membrane excess and refill vesicle pools depleted by exocytosis. To perform these tasks cells can use different endocytosis/recycling mechanisms whose selection will impact on vesicle recycling time and secretion performance. We used FM1-43 to evaluate in the same experiment exocytosis, endocytosis, and recovery of releasable vesicles on mouse chromaffin cells. Various exocytosis levels were induced by a variety of stimuli, and we discriminated the resultant endocytosis-recycling responses according to their ability to rapidly generate releasable vesicles. Exocytosis of Յ20% of plasma membrane (provoked by nicotine/acetylcholine) was followed by total recovery of releasable vesicles. If a stronger stimulus (50 mM K ϩ and 2 mM Ca 2ϩ ) provoking intense exocytosis (51 Ϯ 7%) was applied, endocytosis still retrieved all the fused membrane, but only a fraction (19 Ϯ 2%) was releasable by a second stimulus. Using ADVASEP-7 or bromophenol blue to quickly eliminate fluorescence from noninternalized FM1-43, we determined that this fraction became releasable in Ͻ2 min. The remaining nonreleasable fraction was distributed mainly as fluorescent spots (ϳ0.7 m) selectively labeled by 40-to 70-kDa dextrans and was suppressed by a phosphatidylinositol-3-phosphate kinase inhibitor, suggesting that it had been formed by a bulk retrieval mechanism. We concluded that chromaffin cells can rapidly recycle significant fractions of their total vesicle population, and that this pathway prevails when cholinergic agonists are used as secretagogues. When exocytosis exceeded ϳ20% of plasma membrane, an additional mechanism was activated, which was unable to produce secretory vesicles in our experimental time frame but appeared crucial to maintaining membrane surface homeostasis under extreme conditions. endocytosis; mouse chromaffin cells; calcium signal; FM1-43; ADVASEP-7; bromophenol blue

Excess retrieval is not associated with the cycling of releasable vesicles, but it is related to the formation of non-releasable endosomes. This process is activated by a concerted contribution of Ca(2+) entry through L-channels and Ca(2+) release from endoplasmic reticulum.