Variations in Ca²⁺ Influx Can Alter Chelator-Based Estimates of Ca²⁺ Channel-Synaptic Vesicle Coupling Distance

The timing and probability of synaptic vesicle fusion from presynaptic terminals is governed by the distance between voltage-gated Ca²⁺ channels (VGCCs) and Ca²⁺ sensors for exocytosis. This VGCC-sensor coupling distance can be determined from the fractional block of vesicular release by exogenous Ca²⁺ chelators, which depends on biophysical factors that have not been thoroughly explored. Using numerical simulations of Ca²⁺ reaction and diffusion, as well as vesicular release, we examined the contributions of conductance, density, and open duration of VGCCs, and the influence of endogenous Ca²⁺ buffers on the inhibition of exocytosis by EGTA. We found that estimates of coupling distance are critically influenced by the duration and amplitude of Ca²⁺ influx at active zones, but relatively insensitive to variations of mobile endogenous buffer. High concentrations of EGTA strongly inhibit vesicular release in close proximity (20-30 nm) to VGCCs if the flux duration is brief, but have little influence for longer flux durations that saturate the Ca²⁺ sensor. Therefore, the diversity in presynaptic action potential duration is sufficient to alter EGTA inhibition, resulting in errors potentially as large as 300% if Ca²⁺ entry durations are not considered when estimating VGCC-sensor coupling distances.SIGNIFICANT STATEMENT The coupling distance between voltage-gated Ca²⁺ channels and Ca²⁺ sensors for exocytosis critically determines the timing and probability of neurotransmitter release. Perfusion of presynaptic terminals with the exogenous Ca²⁺ chelator EGTA has been widely used for both qualitative and quantitative estimates of this distance. However, other presynaptic terminal parameters such as the amplitude and duration of Ca²⁺ entry can also influence EGTA inhibition of exocytosis, thus confounding conclusions based on EGTA alone. Here, we performed reaction-diffusion simulations of Ca²⁺-driven synaptic vesicle fusion, which delineate the critical parameters influencing an accurate prediction of coupling distance. Our study provides guidelines for characterizing and understanding how variability in coupling distance across chemical synapses could be estimated accurately.