Cations Residing at the Selectivity Filter of the Voltage-Gated Ca2+-Channel Modify Fusion-Pore Kinetics

Abstract

Strontium (Sr2+), Barium (Ba2+), and Lanthanum (La3+) can substitute for Ca2+ in driving synaptic transmission during membrane depolarization. Ion recognition at the polyglutamate motif (EEEE), comprising the channel selectivity-filter, during voltage-driven transitions, controls the kinetics of the voltage-gated calcium channel (VGCC) and its interactions with the synaptic proteins. We tested the effect of different charge carriers on evoked-release, as a means of exploring the involvement of VGCC in the fusion pore configuration. Employing amperometry recordings in single bovine chromaffin cells we show that the size of the fusion pore, designated by the 'foot'-amplitude, was increased when Ba2+ substituted for Ca2+ and decreased, with La3+. The fusion pore stability, indicated by 'foot'-width, was decreased in La3+. Also, the mean open time of the fusion pore (tfp) was significantly lower in Sr2+ and La3+ compared to Ba2+ and Ca2+. These cations when occupying the selectivity filter reduced the spike frequency in the order of Ca2+ > Sr2+ > Ba2+ > La3+, which is parallel to the reduction in total catecholamine release. The correlation between ion binding at the selectivity filter and fusion pore properties supports a model in which the Ca2+ channel regulates secretion through a site at the selectivity filter, upstream to cation entry into the cell.