Fast exocytosis with few Ca2+ channels in insulin-secreting mouse pancreatic B cells

The association of L-type Ca2+ channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with < 500 alpha (1c) L-type Ca2+ channels, corresponding to a Ca2+ channel density of 0.9 channels per mum(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximate to 650 granules/s) 25 ms after onset of the pulse and is completed within similar to 100 ms. This component represents a subset of approximate to 60 granules situated in the immediate vicinity of the L-type Ca2+ channels, corresponding to similar to 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca2+ revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca2+ concentration of 17 muM, and concentrations > 25 muM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca2+ buffer EGTA but abolished by addition of exogenous LC753-8931 the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca2+, channel, which has been proposed to tether the Ca2+ channels to the secretory granules. In keeping with the idea that secretion is determined by Ca2+ influx through individual Ca2+ channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca2+ channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca2+ from 2.6 to 10 mM. Recordings of single Ca2+ channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca2+ channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca2+ channels as it ensures maximum usage of the Ca2+ entering the cell while minimizing the influence of stochastic variations of the Ca2+ channel activity.