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 M BayK or 0.5 M 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 ␣ 1 1.2 subunit was replaced with the mutant ␣ 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 voltagegated Ca 2؉ channels act as a signaling molecular switch, triggering secretion upstream to ion transport into the cell.The kinetic properties of voltage-gated Ca 2ϩ channels (VGCC) 2 are determined by the conformational changes induced at the channel during membrane depolarization. The kinetics of the L-type VGCC Cav1 is modulated also by allosteric agonists, which include BayK 8644 (BayK), a 1,4-dihydropyridine, FPL64176 (FPL), and CGP 48506 (1-9). BayK, FPL, and CGP 48506, which are structurally unrelated, interact with the cardiac Cav1.2 channel through binding within discrete sites at the transmembrane and extracellular loops of the ␣ 1 1.2 pore-forming subunit (10 -14). BayK enhances macroscopic currents (15) by increasing the rate of transition to "mode 2" single channel behavior (16,17) and the single channel currents by means of lengthening the channel open time (17)(18)(19)(20). BayK binding at selective Cav1.2 regions alters indirectly the selectivity filter, in turn affecting ion permeability (21). The changes observed in channel deactivation and inactivation are sensitive to the type of the cation used as the charge carrier (17).Like BayK 8644 (1), FPL is coupled to the cation binding at the selectivity filter of Cav1.1 acting as an allosteric regulator (22). In neonatal rat ventricular myocytes and rat ventricular cells, FPL enhances Ca 2ϩ influx and slows both the activation and the inactivation kinetics of the Cav1.2 (23). In isolated rat ventricular myocytes, FPL slowed the transition of the channel to a closed or inactivated state (24). In GH3 cells, FPL increased Cav1.2 current amplitude and shifted the current-voltage relationship to negative voltages (25). Single channel analysis showed that FPL increased both the...

Properties of Na+currents conducted by a skeletal muscle L-type Ca2+channel pore mutant (SkEIIIK)

“…32,33 Although Ca v 1.2(L775P) was shown not to conduct inward monovalent (i.e., Li + ) current in the presence of Ca 2+ at relatively hyperpolarized test potentials, 32 entry of the mutant channel into the potentiated (i.e., mode 2) state may alter its permeation characteristics. Thus, while the use of mutant Ca 2+ channels is of value in studying Ca 2+ signaling, it is also important to recognize that such mutant channels may produce inward Na + currents, particularly during prolonged K + -induced depolarizations.…”

From spike to graph—A complete automated single-spike analysis

“…We compared the ability of our method to identify amperometric signals to that of the AmperoStat macro (Hof, 2003) which is widely used (Marom et al, 2007(Marom et al, , 2010 and found that our method identified and analyzed 20% more spikes events especially in nonideal cells with low SNR and baseline change within the experiment (64.8 ± 10.4 vs. 49.2 ± 7.3, paired Student's t-test p < 0.003, Fig. 4E).…”

“…Fig. 4A and B demonstrates two described of these low-pass filters: moving average (Marom et al, 2007(Marom et al, , 2010Yizhar et al, 2004) and Gaussian finite impulse response (FIR) (Mosharov, 2008), compared with our method which does not filter the signal (Fig. 4C).…”

Intra-membrane Signaling Between the Voltage-Gated Ca2+-Channel and Cysteine Residues of Syntaxin 1A Coordinates Synchronous Release

“…Secretion from single vesicles results in amperometric currents represented by spikes. Presentation of a single amperometric event is shown as an amperometric spike and the corresponding foot current48. Total secretion was determined by integrating the area underneath the amperometric spike and presented as picocoulombs (pC).…”