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

S Barg, X Ma, L Eliasson, J Galvanovskis, SO Göpel… - Biophysical journal, 2001 - cell.com
S Barg, X Ma, L Eliasson, J Galvanovskis, SO Göpel, S Obermüller, J Platzer, E Renström
Biophysical journal, 2001cell.com
The association of L-type Ca 2+ 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 Ca 2+
channels, corresponding to a Ca 2+ channel density of 0.9 channels per μm 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 (≈ 650 granules/s) 25ms after onset of …
Abstract
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 α1C 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 1.1pFs−1 (≈650 granules/s) 25ms after onset of the pulse and is completed within ∼100ms. 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 17μM, and concentrations >25μM 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 α1C 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 (15ms) depolarizations was enhanced 2.5-fold by the Ca2+ channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca2+ from 2.6 to 10mM. 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.
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