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

Ca²⁺-dependent K⁺ (BK) channels at varicosities in Xenopus nerve-muscle cell cultures were used to quantify experimentally the instantaneous active zone [Ca²⁺]_AZ resulting from different rates and durations of Ca²⁺ entry in the absence of extrinsic buffers and correlate this with neurotransmitter release. Ca²⁺ tail currents produce mean peak [Ca²⁺]_AZ ~ 30 μM; with continued influx, [Ca²⁺]_AZ reaches ~45-60 μM at different rates depending on Ca²⁺ driving force and duration of influx. Both I_BK and release are dependent on Ca²⁺ 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 Ca²⁺ sensors that trigger release, are located within Ca²⁺ nanodomains while a large fraction of both are located far enough from Ca channels to be blockable by EGTA, (2) the I_BK is more sensitive than the excitatory postsynaptic current (EPSC) to [Ca²⁺]_AZ (K_1/2-26 μM vs. ~36 μM [Ca²⁺]_AZ); (3) with increasing [Ca²⁺]_AZ, the I_BK grows with a Hill coefficient of 2.5, the EPSC with a coefficient of 3.9; (4) release is dependent on the highest [Ca²⁺] 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 [Ca²⁺]_AZ.