Calcium entry into voltage-clamped presynaptic terminals of squid

Voltage-clamp measurements of Ca current and Arsenazo III measurements of intracellular Ca concentration were used to assess Ca ion entry into voltage-clamped presynaptic terminals of squid 'giant' synapses. Depolarization of voltage-clamped terminals filled with Arsenazo III produced absorbance changes consistent with intracellular accumulation of Ca ions. These intracellular Ca transients had a bell-shaped dependence on presynaptic potential and were maximal at approximately -10 mV. Arsenazo III signals recorded from the proximal portion of voltage-clamped presynaptic terminals had a dependence on command potential which was shifted relative to signals recorded from other presynaptic regions. Micro-electrode measurements of presynaptic membrane potential showed that during voltage-clamp depolarizations the proximal region was less depolarized than the rest of the presynaptic terminal. This indicates that voltage-clamped presynaptic terminals may be poorly controlled at their proximal region due to current flow into the adjacent axon. This poor control can cause heterogeneous Ca entry into the presynaptic terminal and thus heterogeneous release of transmitter along the terminal. Application of Ca ions from an extracellular pipette positioned near the distal end of the presynaptic terminal was used to restrict Ca entry to this well-controlled region. Local Ca application decreased the contribution of release from the poorly controlled proximal region to synaptic transfer curves. Presynaptic Ca currents were derived by correcting membrane currents for leakage and capacitive currents and other currents measured in the absence of Ca application. Ca currents measured in this way activated along a sigmoidal time course and did not inactivate for depolarizations as long as 25 ms. Peak Ca currents occurred at approximately -10 mV and inward Ca currents had an apparent 'reversal potential' near +60 mV. Ca channel activation, assessed with tail current measurements, was half-maximal at -13 mV and maximal at +20 mV. Simultaneous measurements of presynaptic Ca currents and Arsenazo III transients revealed a quantitative correspondence between Ca current integrals and Arsenazo III signal amplitude. This suggests that both methods provide reliable measures of Ca ion entry into presynaptic terminals under these conditions.