Conformational changes induced in voltage-gated calcium channel Cav1.2 by BayK 8644 or FPL64176 modify the kinetics of secretion independently of Ca2+ influx

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.

FIGURE 1.

BayK-modified spike frequency as monitored by amperometry in chromaffin cells. A, amperometry currents were triggered by K60 (see “Experimental Procedures”) in the presence or absence of 2 μm BayK, using Ca²⁺ as the charge carrier. No amperometric currents were detected in the absence of K60 (top inset). Amperometric currents were blocked ∼90% in the presence of 5 μm nifedipine (bottom inset). B, left panel, spike frequency. Cumulative events per cell, averaged for cells with (n = 29) or without (n = 37) 2 μm BayK, were plotted versus time. Right panel, expanded view of the initial cumulative spike counts (upper panel) and sustained cumulative spike count (lower panel). The mean frequency of the initial rate was calculated as the maximum slope in plot B during the first 20 s of K60 stimulation (upper panel) (Table 1). The mean frequency of the sustained rate of secretion was calculated as the maximum slope during the time period of 60–180 s (lower panel). C and D, plots similar to those in A and B except that 2 mm Ba²⁺ (26 cells without and 30 cells with BayK) was used as the charge carrier. E and F, plots similar to A and B except for 0.2 mm La³⁺ (30 cells without and 21 cells with BayK). Means were calculated for individual cells as an average of more than 500 spike events.

FIGURE 2.

Effect of BayK in Ca²⁺, Ba²⁺, or La³⁺ on foot amplitude in single chromaffin cells. A, presentation of single amperometric event kinetic properties of spikes. Peak amplitude, half-width, 50–90% rise time, and integrated spike (Q, gray area) are shown. Foot signal, foot width, foot amplitude, and integrated foot (Q, black area) are shown. Amperometric currents were triggered by a 30-s pulse of K60 from single cells. B, amperometric recording of the fusion of a single vesicle with or without 2 μm BayK in 2 mm Ca²⁺. B, right panel, cumulative distribution plot of foot number and analysis of foot amplitude in Ca²⁺. Inset, mean values for foot amplitude with or without BayK, n = 501, n = 911 events per data point, respectively. C, amperometric recording of the fusion of a single vesicle with or without 2 μm BayK in 2 mm Ba²⁺. C, right panel, data plotted in similar fashion as in B, right panel, in 2 mm Ba²⁺, n = 658; n = 494, respectively. D, amperometric recording of single vesicle fusion with or without 2 μm BayK in 0.2 mm La³⁺. D, right panel, data obtained in 0.2 mm La³⁺ plotted similar to B, right panel, with (n = 625) or without 2 μm BayK (n = 429). *, p < 0.05; ***, p < 0.001.

FIGURE 3.

FPL-modified spike frequency in chromaffin cells. A, spike frequency (left panel). Cumulative events per cell, averaged for control or 0.5 μm FPL-treated cells, were plotted versus time; right panel, an expanded view of the initial cumulative spike counts (upper panel) and sustained cumulative spike counts (lower panel). The mean frequency of the initial rate was calculated as the maximum slope in plot B during the first 20 s of K60 stimulation (upper panel) (Table 1) and the mean frequency of the sustained rate of secretion during the time period of 60–180 s (lower panel). C and E, amperometry currents were triggered by K60 in the presence and in the absence of 0.5 μm FPL, using Ba²⁺ (C) or La³⁺ (E) as the charge carrier. D, plots similar to those in B with 2 mm Ba²⁺ and in F, with 0.2 mm La³⁺.

FIGURE 4.

FPL modifies foot amplitude in single chromaffin cells in Ca²⁺, Ba²⁺, or La³⁺. Amperometric currents were triggered by a 30-s pulse of K60 from single cells in solutions of 2 mm Ca²⁺, 2 mm Ba²⁺, or 0.2 mm La³⁺ with or without 0.5 μm FPL. A, representative foot in Ca²⁺, Ba²⁺, or La³⁺ elicited with and without 0.5 μm FPL; bars are as indicated. B, cumulative distribution plot of foot events and analysis of foot amplitude. Insets, mean values of foot amplitude with or without 0.5 μm FPL, see supplemental Table SII. *, p < 0.05; ***, p < 0.001. The number of foot amplitudes analyzed is given in supplemental Table SII.

FIGURE 5.

FPL affects depolarization-evoked release mediated in cells infected with the mutated Cav1.2 channel subunit pSFV α₁1.2/L775P. A, amperometric currents were triggered by a 10-s pulse of K60 from single cells infected with either α₁1.2 subunit or the impermeable α₁1.2/L775P with 2 mm Ca²⁺ as the charge carrier, with or without 0.5 μm FPL and in the presence of 5 μm nifedipine. B, cumulative distribution of spikes plotted versus time after the onset of K60 depolarization in cells infected with WT α₁1.2 subunit (left panel) and the mutated α₁1.2/L775P subunit (right panel) in the presence (●, ♦) and in the absence of 0.5 μm FPL (○, ◇), respectively. Inset, expanded scale of the cumulative events elicited by a 10-s pulse of K60 from single cells infected with either α₁1.2 Cav1.2 subunit (left panel) or the impermeable α₁1.2/L775P (right panel) with 2 mm Ca²⁺ as the charge carrier, emphasizing the initial rates (10–30 s) and sustained rates (30–60 s).

FIGURE 6.

Initial and sustained rates of secretion mediated by α₁1.2 and α₁1.2/L775P are elevated to a similar extent by FPL. Initial (10–30 s) (A) and sustained (60–180 s) (B) slopes of the corresponding cumulative spike plot (see “Experimental Procedures”) of α₁1.2 and α₁1.2/L775P in the presence and absence of FPL. The extent of FPL effect is shown by the ratio of the initial (A) and sustained rates (B) with and without FPL (right).

FIGURE 7.

Effect of FPL on release mediated by a Ca²⁺-impermeable channel. Amperometric currents were triggered by a 10-s pulse of K60 from single cells infected with either WT α₁1.2 subunit (left) or the impermeable α₁1.2/L775P (right) with 2 mm Ca²⁺ as the charge carrier, with or without 0.5 μm FPL and in the presence of 5 μm nifedipine. A, representative foot traces of single vesicle release events. B, cumulative distribution of foot events mediated by α₁1.2 WT subunit (left panel) and α₁1.2/L775P (right panel) plotted versus time after the onset of K60 depolarization. Inset, analysis of foot amplitude, mean values for foot amplitude events (see supplemental Table SIV). C, cumulative distribution of foot events mediated by α₁1.2 WT subunit (left panel) and α₁1.2L/775P (right panel) plotted versus time after the onset of K60 depolarization; inset, analysis of foot width. D, single exponential fits to time distributions yielded the mean fusion pore open time (τ_fp, left panel); inset, τ_fp values (right panel). *, p < 0.05.

FIGURE 8.

Schematic view of FPL-modified α₁1.2 subunit. The binding of FPL to the α₁1.2 subunit of the channel modified the channel properties and increased Ca²⁺ entry (left panel). The α₁1.2/L775P subunit is similarly modified by FPL but does not conduct Ca²⁺ (right panel). The similar modulation of secretion by the WT α₁1.2 and the mutated channel α₁1.2/L775P suggests the involvement of structural changes induced by FPL during voltage perturbation and the subsequent calcium ion occupancy of the selectivity filter prior to Ca²⁺ influx (40).