In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.

Activation of calcium-ion (

Potassium-stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K' in the presence of 2 mM Ca2+ for 10 s, applied at 5-min intervals) was inhibited by the dihydropyridine furnidipine (3 PM) by 50%. o-Conotoxin MVIIC (CTx-MVIIC, 3 PM) also reduced the secretory response by about half. Combined CTx-MVIIC plus furnidipine blocked 100% catecholamine release. "sCa*+ uptake and cytosolic Car' concentrations ([Ca"],) in K'-depolarized cells were partially blocked by furnidipine or CTx-MVIIC, and completely inhibited by both agents. The whole cell current through Ca" channels carried by Ba*' (Ia.) was partially blocked by CTx-MVIIC. Although w-conotoxin GVIA (CTx-GVIA, I PM) and w-agatoxin IVA (Aga-IVA, 0.2 PM) partially inhibited 45CaZ' entry, I,, and the increase in [Ca"],, the combination of both toxins did not affect the K'-evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q-like Ca*' channel which has a prominent role in controlling exocytosis. They also suggest that Q-and L-type Car' channels, but not N-or P-types are localized near exocytotic active sites in the plasmalemma.

Patch-clamp measurements of Ca 2ϩ currents and membrane capacitance were performed on slices of mouse adrenal glands, using the perforated-patch configuration of the patch-clamp technique. These recording conditions are much closer to the in vivo situation than those used so far in most electrophysiological studies in adrenal chromaffin cells (isolated cells maintained in culture and whole-cell configuration). We observed profound discrepancies in the quantities of Ca 2ϩ channel subtypes (P-, Q-, N-, and L-type Ca 2ϩ channels) described for isolated mouse chromaffin cells maintained in culture. Differences with respect to previous studies may be attributable not only to culture conditions, but also to the patch-clamp configuration used. Our experiments revealed the presence of a Ca 2ϩ channel subtype never before described in chromaffin cells, a toxin and dihydropyridine-resistant Ca 2ϩ channel with fast inactivation kinetics, similar to the R-type Ca 2ϩ channel described in neurons. This channel contributes 22% to the total Ca 2ϩ current and controls 55% of the rapid secretory response evoked by short depolarizing pulses. Our results indicate that R-type Ca 2ϩ channels are in close proximity with the exocytotic machinery to rapidly regulate the secretory process.