NMDA-mediated modulation of γ-aminobutyric acid type A receptor function in cerebellar granule neurons

Abstract

GABA(A) receptors are ligand-gated Cl ion channels with multiple clinically relevant drug-recognition sites. We have previously shown that stimulation of N-methyl-D-aspartic acid (NMDA)-specific glutamate receptors quantitatively alters selected GABA(A) receptor subunit mRNAs and proteins in primary cultures of rat cerebellar granule neurons. We used whole-cell recordings of GABA-elicited Cl currents and flunitrazepam binding experiments in granule cell cultures maintained in low K+ (12.5 mM), cells maintained in low K+ and treated with a single dose of NMDA (10 μM), and cell cultures maintained in depolarizing concentrations of K+ (25 mM). The EC50 obtained from the dose-response curves for GABA in eliciting a maximal response was comparable in neurons maintained in high K+ or in low K+ and treated with a single dose of NMDA, but that it increased significantly in cells maintained in low K+. The potentiation of GABA-gated Cl currents by flunitrazepam increased significantly, while the negative allosteric modulator methyl-6,7- dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) was significantly more effective in cultures either maintained in high K+ or treated with NMDA. This was coincident with a twofold increase in the B(max) associated with flunitrazepam binding. To further characterize the receptor assemblies present in the depolarization and NMDA induced paradigms, the Zn2+-induced inhibition of GABA-gated Cl currents was reduced as was the inhibition mediated by furosemide. Our data indicate that GABA(A) receptor assemblies alter their composition in response to excitatory afferent receptor stimulation.