Abstract
The role of the L-type calcium channel (Cav1.2) as a molecular switch that triggers secretion prior to Ca(2+) transport has previously been demonstrated in bovine chromaffin cells and rat pancreatic beta cells. Here, we examined the effect of specific Cav1.2 allosteric modulators, BayK 8644 (BayK) and FPL64176 (FPL), on the kinetics of catecholamine release, as monitored by amperometry in single bovine chromaffin cells. We show that 2 microm BayK or 0.5 microm FPL accelerates the rate of catecholamine secretion to a similar extent in the presence either of the permeable Ca(2+) and Ba(2+) or the impermeable charge carrier La(3+). These results suggest that structural rearrangements generated through the binding of BayK or FPL, by altering the channel activity, could affect depolarization-evoked secretion prior to cation transport. FPL also accelerated the rate of secretion mediated by a Ca(2+)-impermeable channel made by replacing the wild type alpha(1)1.2 subunit was replaced with the mutant alpha(1)1.2/L775P. Furthermore, BayK and FPL modified the kinetic parameters of the fusion pore formation, which represent the initial contact between the vesicle lumen and the extracellular medium. A direct link between the channel activity and evoked secretion lends additional support to the view that the voltage-gated Ca(2+) channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.
Highlights
The kinetic properties of voltage-gated Ca2ϩ channels (VGCC)2 are determined by the conformational changes induced at the channel during membrane depolarization
Corroborating with the proposed model, secretion could be triggered by membrane depolarization in chromaffin cells where the normal ␣11.2 pore-forming subunit of Cav1.2 was replaced by a Ca2ϩ-impermeable subunit ␣11.2/L775P [40]
We have explored this concept further, testing whether the mutually exclusive structural rearrangements conferred upon the channel by BayK or FPL could affect the kinetics of secretion in bovine chromaffin cells, prior to and independently of cation influx
Summary
Materials—BayK 8644, 1,4-dihydro-2,6-dimethyl-5-nitro-4(2(trifluoromethyl)phenyl)pyridine-3-carboxylic acid methyl ester, was from Sigma. FPL in 0.0001% DMSO was used at a final concentration of 0.5 M. Nifedipine in 0.001% DMSO was used at a final concentration of 5 M (Sigma). In a separate control experiment, 0.02– 0.2% DMSO had no effect on secretion in chromaffin cells. Semliki Forest Virus Infection—Semliki Forest viruses of ␣11.2 (D1733) and ␣11.2/L775P mutant [41] were prepared, and chromaffin cells were infected as described previously [40]. La3ϩ-mediated secretion in chromaffin cells is optimal at a concentration in the range of 0.1– 0.2 mM for this trivalent ion [31]. The beginning of the foot was defined as the time when the signal exceeded the noise. Spikes exceeding three times the background noise (Ͼ10 pA) were analyzed. The number of foot analyzed is shown in supplemental Table SII
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