Presynaptic Calcium Channels and the Depletion of Synaptic Cleft Calcium Ions

Abstract

The entry of calcium ions (Ca2+) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca2+ into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca2+ depletion. The terminal was voltage clamped at a holding potential ( V H) of −80 mV and a depolarizing pulse was applied to a range of potentials (−60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V H, by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca2+electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V 1/2 of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V 1/2 of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba2+ is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca2+ depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.