The effect of intracellular Ca2+ on GABA-activated currents in cerebellar granule cells in culture

The patch clamp technique was used to study the effects of intracellular free calcium ([Ca2+]i) on GABAA-evoked whole-cell and single channel currents of cultured cerebellar granule cells. Changes in [Ca2+]i were obtained by adding to the extracellular solution the calcium ionophore A23187 (2 μm). The relationship between [Ca2+]i and [Ca2+]O in the presence or absence of A23187 was assessed using fluorimetric measurements from Fura-2 loaded cells. In 2 m m [Ca2+]o and A23187, [Ca2+]i was about 1.5 μm, whereas in the absence of A23187 it was about 250 n m. In whole-cell experiments (symmetrical chloride concentrations) at -50 mV, GABA (0.5 μm) evoked inward currents that did not desensitize. Bath application of A23187 significantly reduced the steady-state amplitude of GABA currents by 37 ± 6%. Single channel currents activated by GABA (0.5 μm) were also recorded in the outside-out configuration of the patch clamp technique. Kinetic analysis of single channel events revealed that A23187 significantly increased the long closed time constant (τc3) without affecting the open time constants (τo1 and τo2) or the short and medium closed time constants (τc1 and τc2). Moreover, application of A23187 induced a significant reduction of burst duration (τb). We conclude that a rise in [Ca2+]i by A23187 may decrease the binding affinity of GABA for the GABAA receptor. We thank Prof. D. Colquhoun for critical reading of the manuscript and Drs. F. Vittur and M. Fragonas for allowing us the use of the spectrofluorimeter. © 1994 Springer-Verlag New York Inc.