Ca²⁺ buffering at a drosophila larval synaptic terminal

A quantitative analysis of Ca²⁺ dynamics requires knowledge of the Ca²⁺-binding ratio (κ_S); this has not been measured at Drosophila synaptic terminals or any invertebrate synaptic terminal. We measured κ_S at a Ib motor terminal in Drosophila larvae comparing single-AP Ca²⁺ transients in synaptic terminals that contained varying concentrations of the Ca²⁺ indicator, Oregon Green 488 BAPTA-1 (OGB-1). Using a linear single-compartment model, κ_S was calculated based upon the effect of [OGB-1] on the time constant (τ_decay) for the decay of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). This gave a κ_S of 77 indicating that nearly 99% of entering Ca²⁺ is immediately bound by endogenous fast Ca²⁺ buffers. Extrapolation to zero [OGB-1] gave a τ_decay of 46 ms and a Ca²⁺-removal rate constant of 1641 s^-1 for single APs. We calculated that a single AP produced an increase in [Ca²⁺]_i of 196 nM and an increase in the total intracellular [Ca²⁺](free + bound) of 15.3 μM for measurements made in 1.0 mM external Ca²⁺. The increase in [Ca²⁺]_i for AP trains was 185 nM/ 10 Hz; this gave a Ca²⁺ extrusion rate constant of 827 s^-1, which likely reflects the activity of the plasma membrane Ca²⁺ ATPase. Experiments were performed to examine the effect of altering external Ca²⁺ or Mg²⁺ on Ca²⁺ influx at these terminals.