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

Abstract

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 microM). 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 mM [Ca2+]o and A23187, [Ca2+]i was about 1.5 microM, whereas in the absence of A23187 it was about 250 nM. In whole-cell experiments (symmetrical chloride concentrations) at -50 mV, GABA (0.5 microM) 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 microM) 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 (tau c3) without affecting the open time constants (tau o1 and tau o2) or the short and medium closed time constants (tau c1 and tau c2). Moreover, application of A23187 induced a significant reduction of burst duration (tau b). We conclude that a rise in [Ca2+]i by A23187 may decrease the binding affinity of GABA for the GABAA receptor.