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 α1_c L-type Ca²⁺ channels, corresponding to a Ca²⁺ channel density of 0.9 channels per μm². Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs^-1 (≈650 granules/s) 25 ms after onset of the pulse and is completed within ∼100 ms. This component represents a subset of ≈60 granules situated in the immediate vicinity of the L-type Ca²⁺ channels, corresponding 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 μM, and concentrations >25 μM 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 L_c753-893, the 140 amino acids of the cytoplasmic loop connecting the 2nd and 3rd transmembrane region of the α1_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.