Calcium gradients and exocytosis in bovine adrenal chromaffin cells

The relationship between the localized Ca2+ concentration and depolarization-induced exocytosis was studied in patch-clamped adrenal chromaffin cells using pulsed-laser Ca2+ imaging and membrane capacitance measurements. Short depolarizing voltage steps induced Ca2+ gradients and small “synchronous” increases in capacitance during the pulses. Longer pulses increased the capacitance changes, which saturated at 16 fF, suggesting the presence of a small immediately releasable pool of fusion-ready vesicles. A Hill plot of the capacitance changes versus the estimated Ca2+ concentration in a thin (100 nm) shell beneath the membrane gave n = 2.3 and Kd = 1.4 μM. Repetitive stimulation elicited a more complex pattern of exocytosis: early pulses induced synchronous capacitance increases, but after five or more pulses there was facilitation of the synchronous responses and gradual increases in capacitance continued between pulses (asynchronous exocytosis) as the steep submembrane Ca2+ gradients collapsed. Raising the pipette Ca2+ concentration led to early facilitation of the synchronous response and early appearance of asynchronous exocytosis. We used this data to develop a kinetic model of depolarization-induced exocytosis, where Ca2+-dependent fusion of vesicles occurs from a small immediately releasable pool with an affinity of 1-2 μM and vesicles are mobilized to this pool in a Ca2+-dependent manner.