Experimentally monitored calcium dynamics at synaptic active zones during neurotransmitter release in neuron-muscle cell cultures.

Ca-dependent K (BK) channels at varicosities in Xenopus nerve-muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca] resulting from different rates and durations of Ca entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca tail currents produce mean peak [Ca] ~ 30 μM; with continued influx, [Ca] reaches ~45-60 μM at different rates depending on Ca driving force and duration of influx. Both I and release are dependent on Ca microdomains composed of both N- and L-type Ca channels. Domains collapse with a time constant of ~0.6 ms. We have constructed an active zone (AZ) model that approximately fits this data, and depends on incorporation of the high-capacity, low-affinity fixed buffer represented by phospholipid charges in the plasma membrane. Our observations suggest that in this preparation, (1) some BK channels, but few if any of the Ca sensors that trigger release, are located within Ca nanodomains while a large fraction of both are located far enough from Ca channels to be blockable by EGTA, (2) the I is more sensitive than the excitatory postsynaptic current (EPSC) to [Ca] (K-26 μM vs. ~36 μM [Ca]); (3) with increasing [Ca], the I grows with a Hill coefficient of 2.5, the EPSC with a coefficient of 3.9; (4) release is dependent on the highest [Ca] achieved, independent of the time to reach it; (5) the varicosity synapses differ from mature frog nmjs in significant ways; and (6) BK channels are useful reporters of local [Ca].