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Biophys J, December 2001, p. 3308-3323, Vol. 81, No. 6

Fast Exocytosis with Few Ca2+ Channels in Insulin-Secreting Mouse Pancreatic B Cells

Sebastian Barg,* Xiaosong Ma,* Lena Eliasson,* Juris Galvanovskis,* Sven O. Göpel,* Stefanie Obermüller,* Josef Platzer,dagger Erik Renström,* Michel Trus,Dagger Daphne Atlas,Dagger Jörg Striessnig,dagger and Patrik Rorsman*

 *Department of Molecular and Cellular Physiology, Institute of Physiological Sciences, Lund University, BMC F11, SE-221 84 Lund, Sweden;  dagger Institut für Biochemische Pharmakologie, Peter-Mayrstrasse 1, A-6020 Innsbruck, Austria; and  Dagger Department of Biological Chemistry, The Hebrew University of Jerusalem, 91904 Israel

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 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 (approx 650 granules/s) 25 ms after onset of the pulse and is completed within ~100 ms. This component represents a subset of approx 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 LC753-893, the 140 amino acids of the cytoplasmic loop connecting the 2nd and 3rd transmembrane region of the alpha 1C 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.

Biophys J, December 2001, p. 3308-3323, Vol. 81, No. 6
© 2001 by the Biophysical Society   0006-3495/01/12/3308/16  $2.00


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