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

Abstract

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 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(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) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM 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 Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Mathematical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in β-cells from mice and humans

“…For this reason, depolarizations of different durations are often applied to follow the dynamics of the exocytotic response, assuming that the kinetics of exocytosis is unchanged from pulse to pulse (Ammälä et al, 1993b;Horrigan and Bookman, 1994;Barg et al, 2001;Braun et al, 2008).…”

“…In single mouse β-cells, the exocytotic rate is higher for shorter than for longer pulses (Ammälä et al, 1993b;Barg et al, 2001;Eliasson et al, 2003). The drop in the exocytotic rate at sustained depolarizations has been suggested to result either from Ca 2+ -channel inactivation (Ammälä et al, 1993b;Engisch and Nowycky, 1996) or depletion of a finite pool of immediately releasable granules (Barg et al, 2001;Eliasson et al, 2003).…”

“…The drop in the exocytotic rate at sustained depolarizations has been suggested to result either from Ca 2+ -channel inactivation (Ammälä et al, 1993b;Engisch and Nowycky, 1996) or depletion of a finite pool of immediately releasable granules (Barg et al, 2001;Eliasson et al, 2003). A frequently applied method to relate the evoked exocytotic response and the Ca 2+ -current, is to plot ∆C m as a function of the integrated current, i.e.…”

“…A frequently applied method to relate the evoked exocytotic response and the Ca 2+ -current, is to plot ∆C m as a function of the integrated current, i.e. the charge Q that entered during the depolarization (Engisch and Nowycky, 1996;Barg et al, 2001;Eliasson et al, 2003;Vikman et al, 2006).…”

“…A relative large submembrane compartment consisting of a shell of depth ∼400 nm was used based on Klingauf and Neher (1997), who showed that in the case of sparse Ca 2+ -channels, the dynamics between channels is corresponds to a shell model at a depth of ∼ 23% of the channel distance. In mouse β-cells the interchannel distance has been estimated to be 1.2 µm (Barg et al, 2001). The value of B was based on v mem and v cell following De Schutter and Smolen (1998), and adjusted slightly to optimize the model behavior.…”