Modulation of Ca2+ Influx at Hyperpolarized Membrane Potentials Alters Depolarization-Triggered Exocytosis in Bovine Chromaffin Cells

Abstract

Perturbations in Ca2+ entry via neuronal transient receptor potential (TRP) and ORAI family proteins have been shown to alter synaptic signaling and architecture, and suggested to play a role in a spectrum of neuropathological conditions. Specific mechanisms by which of TRP/ORAI channels modulate synaptic transmission are not completely understood. Here we employed a combination of imaging and electrophysiological techniques to monitor changes in intracellular Ca2+ concentration ([Ca2+]i), membrane currents at hyperpolarized membrane potentials, voltage-gated Ca2+ currents, and changes in membrane capacitance to investigate effects of modulation of Ca2+ entry at hyperpolarized membrane potentials on exocytosis in bovine chromaffin cells. A small inwardly directed membrane current (I hold), whose amplitude was dependent on the concentration of extracellular Ca2+ and Na+, was present at resting membrane potential. Augmentation of I hold either by hyperpolarization (from −50 mV to −90 mV) or stimulation with bradykinin (1 μM), a catecholamine secretagogue, elevated [Ca2+]i at hyperpolarized potentials and facilitated exocytotic responses triggered by short depolarizations (<500 ms in duration). Exocytotic responses triggered by long depolarizations (>500 ms in duration) were smaller at −90 mV holding potential compared with those at −50 mV holding potential. Reversal of Na+/Ca2+ exchanger by removal of Na+ from bath solution did not cause depolarization-independent exocytosis at −50 mV holding potential. Membrane hyperpolarization from −50 mV to −90 mV in Na+ -free bath solution evoked depolarization-independent exocytosis with the maximal release rate of about 6 fF/s. Our findings provide first direct evidence that 1) Ca2+ influx via native Ca2+ channels operating at negative voltages is functionally coupled with Na+/Ca2+ exchanger and that 2) perturbation in Ca2+ influx an negative voltages with physiologically relevant stimuli significantly modulate exocytotic responses to depolarization-triggered Ca2+ influx in neuroendocrine cells.