THE EFFECT OF INTRACELLULAR CA2-ACTIVATED CURRENTS IN CEREBELLAR GRANULE CELLS IN CULTURE( ON GABA)

The patch clamp technique was used to study the effects of intracellul ar free calcium ([Ca2+](i)) on GABA(A)-evoked whole-cell and single ch annel currents of cultured cerebellar granule cells. Changes in [Ca2+] (i) were obtained by adding to the extracellular solution the calcium ionophore A23187 (2 mu M). The relationship between [Ca2+](i) and [Ca2 +](o) in the presence or absence of A23187 was assessed using fluorime tric measurements from Fura-2 loaded cells. In 2 mM [Ca2+](o) and A231 87, [Ca2+]i was about 1.5 mu M, whereas in the absence of A23187 it wa s about 250 nM. In whole-cell experiments (symmetrical chloride concen trations) at -50 mV, GABA (0.5 mu M) evoked inward currents that did n ot desensitize. Bath application of A23187 significantly reduced the s teady-state amplitude of GABA currents by 37 +/- 6%. Single channel cu rrents activated by GABA (0.5 mu M) were also recorded in the outside- out configuration of the patch clamp technique. Kinetic analysis of si ngle channel events revealed that A23187 significantly increased the l ong closed time constant (tau(c3)) without affecting the open time con stants (tau(o1) and tau(o2)) or the short and medium closed time const ants (tau(c1) and tau(c2)). Moreover, application of A23187 induced a significant reduction of burst duration (tau(b)). We conclude that a r ise in [Ca2+](i) by A23187 may decrease the binding affinity of GABA f or the GABA(A) receptor.