Docked granules, the exocytic burst, and the need for ATP hydrolysis in endocrine cells

Abstract

Ca(2+)-triggered exocytosis was studied in single rat melanotrophs and bovine chromaffin cells by capacitance measurements. Sustained exocytosis required MgATP, but even in the absence of MgATP, Ca2+ could trigger exocytosis of 2700 granules in a typical melanotroph and of 840 granules in a chromaffin cell. Granules undergoing ATP-independent exocytosis were similar in number to those appearing docked to the plasmalemma in quickly frozen unfixed sections (3300 in a melanotroph and 830 in a chromaffin cell). Most exocytosis required tens of seconds, but a small pool of granules was released in tens of milliseconds. Evidently, only a small subset of docked granules is rapidly releasable. We suggest that, temporally, the last ATP-dependent step in exocytosis is closely associated with docking and that docked granules reach fusion competence only after subsequent steps.

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…Thus, changes are most consistent with isolation of a reduced pool of fusion-competent CV from LY29-and Wrt-treated eggs rather than with a direct role of PIP in triggered fusion. Reversible priming steps exist in the fully docked state; PI-3K activity seems likely to define the last necessary priming step in the docked state, consistent with earlier findings that only~10% of docked neuroendocrine vesicles were fully release-ready, the remainder having to pass rapidly through a final preparatory step to confer fusion competence [66].…”

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…This is potentially of importance since it has been suggested that SNARE proteins of docked vesicles would not be accessible to neurotoxins [38]. It has been suggested that release of docked granules corresponds to that component of release that is MgATP-independent [39]. Release of catecholamine due to GppNHp was, however, reduced by about 50% in the absence of ATP, as is the case for Ca 2÷-induced release (Fig.…”

“…The release due to GppNHp was 2.5 _+ 0.2% of total cellular catecholamine above basal from 17 cell batches. This extent of release could conceivably be accounted for by release of only docked secretory granules (corresponding to 1.5-3% of total granules per cell [15,39]). This is potentially of importance since it has been suggested that SNARE proteins of docked vesicles would not be accessible to neurotoxins [38].…”

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

“…In B-cells, the size of the burst that can be released immediately upon Ca 2π -influx corresponds to less than 1% of the total number of granules (Barg et al 2001a(Barg et al , 2002a. Refilling of this functionally defined ''readily releasable pool'' is ATP-, Ca 2π -and temperature-dependent (Bittner & Holz 1992;Parsons et al 1995, Renström et al 1996a& b, Eliasson et al 1997Gromada et al 1999), and commonly referred to as ''priming'' (Hay & Martin 1992). In the B-cells, the ''readily releasable pool'' can be depleted in less than a second (Barg et al 2001a(Barg et al & 2002a, whereas its refilling takes up to a minute Barg et al 2002a).…”

“…Although capacitance measurements monitor only membrane fusion, kinetic analysis of the capacitance MiniReview data allows limited predictions of processes that precede exocytosis, e.g., the rate at which granules become readily releasable (Parsons et al 1995). During trains of depolarizations, exocytosis proceeds throughout the train, with the fastest rate during the first depolarization and progressively smaller responses during subsequent depolarizations ( fig.…”