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Figure S2. Exocytosis (A) and Ca²⁺ influx (B) elicited by depolarizations (-89 to 1 mV) of progressively longer duration to estimate the size of IRP and derive suitable double-pulse protocol parameters (dashed lines, pulse durations used in Fig. 4). Up to a pulse duration of 250 ms, the Ca²⁺ charge entry increased almost linearly with the length of the depolarization (B). The exocytotic ΔC_m, however, increased rapidly and showed saturation already around 200 ms (A). The increase in ΔC_m up to that point could be fitted with a single exponential function that approached 16 fF with a time constant of 40 ms. The depression of ΔC_m can be interpreted as the fast depletion of a release ready pool. The experiment was performed with [Ca²⁺]_o = 7 mM, pulse durations have been randomized and pauses of at least 30 s were given between stimulations to allow for pool refilling.