GABA-induced uncoupling of GABA/benzodiazepine site interactions is associated with increased phosphorylation of the GABAA receptor.

The use-dependent regulation of the GABAA receptor occurs under physiological, pathological, and pharmacological conditions. Tolerance induced by prolonged administration of benzodiazepines is associated with changes in GABAA receptor function. Chronic exposure of neurons to GABA for 48 hr induces a downregulation of the GABAA receptor number and an uncoupling of the GABA/benzodiazepine site interactions. A single brief exposure ((t1/2) = 3 min) of rat neocortical neurons to the neurotransmitter initiates a process that results in uncoupling hours later (t(1/2) = 12 hr) without alterations in the number of GABAA receptors and provides a paradigm to study the uncoupling mechanism selectively. Here we report that uncoupling induced by a brief GABAA receptor activation is blocked by the coincubation with inhibitors of protein kinases A and C, indicating that the uncoupling is mediated by the activation of a phosphorylation cascade. GABA-induced uncoupling is accompanied by subunit-selective changes in the GABAA receptor mRNA levels. However, the GABA-induced downregulation of the α3 subunit mRNA level is not altered by the kinase inhibitors, suggesting that the uncoupling is the result of a posttranscriptional regulatory process. GABA exposure also produces an increase in the serine phosphorylation on the GABAA receptor γ2 subunit. Taken together, our results suggest that the GABA-induced uncoupling is mediated by a posttranscriptional mechanism involving an increase in the phosphorylation of GABAA receptors. The uncoupling of the GABAA receptor may represent a compensatory mechanism to control GABAergic neurotransmission under conditions in which receptors are persistently activated.

Pharmacological Properties of DOV 315,090, an ocinaplon metabolite.

BACKGROUND: Compounds targeting the benzodiazepine binding site of the GABAA-R are widely prescribed for the treatment of anxiety disorders, epilepsy, and insomnia as well as for pre-anesthetic sedation and muscle relaxation. It has been hypothesized that these various pharmacological effects are mediated by different GABAA-R subtypes. If this hypothesis is correct, then it may be possible to develop compounds targeting particular GABAA-R subtypes as, for example, selective anxiolytics with a diminished side effect profile. The pyrazolo[1,5-a]-pyrimidine ocinaplon is anxioselective in both preclinical studies and in patients with generalized anxiety disorder, but does not exhibit the selectivity between alpha1/alpha2-containing receptors for an anxioselective that is predicted by studies using transgenic mice. RESULTS: We hypothesized that the pharmacological properties of ocinaplon in vivo might be influenced by an active biotransformation product with greater selectivity for the alpha2 subunit relative to alpha1. One hour after administration of ocinaplon, the plasma concentration of its primary biotransformation product, DOV 315,090, is 38% of the parent compound. The pharmacological properties of DOV 315,090 were assessed using radioligand binding studies and two-electrode voltage clamp electrophysiology. We report that DOV 315,090 possesses modulatory activity at GABAA-Rs, but that its selectivity profile is similar to that of ocinaplon. CONCLUSION: These findings imply that DOV 315,090 could contribute to the action of ocinaplon in vivo, but that the anxioselective properties of ocinaplon cannot be readily explained by a subtype selective effect/action of DOV 315,090. Further inquiry is required to identify the extent to which different subtypes are involved in the anxiolytic and other pharmacological effects of GABAA-R modulators.

Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor.

The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers.

NMDA receptors contribute to the survival promoting effect of high potassium in cultured cerebellar granule cells.

The present report further analyzes the survival promoting effect of high potassium, a condition that mimics neural activity in cultured cerebellar granule cells, an excellent model to study trophic mechanisms induced by depolarization and trophic factors. We found that the survival promoting effect measured at 7 days in vitro (DIV 7) of depolarizing potassium concentrations (25 mM KCl), added at DIV2, is partially prevented by adding at DIV 2 the non-competitive NMDA blocker MK801 (10 microM). The concentration of MK801 used blocks completely the survival promoting effect of a supramaximal effective concentration of NMDA (100 microM). The addition at DIV 2 of anti-brain derived neurotrophic factor (anti-BDNF) antibody, failed to modify the effect of high potassium. The present report provides evidences that in cultured cerebellar granule cells, high potassium-induced survival promoting effect is due in part by the activation of NMDA receptors. The effect does not require the presence of BDNF.

GABA induces activity dependent delayed-onset uncoupling of GABA/benzodiazepine site interactions in neocortical neurons.

Changes in the function of type A gamma-aminobutyric acid receptors (GABA(A)Rs) are associated with neuronal development and tolerance to the sedative-hypnotic effects of GABA(A)R positive modulators. Persistent activation of GABA(A)Rs by millimolar concentrations of GABA occurs under physiological conditions as GABAergic fast-spiking neurons in neocortex and cerebellum exhibit basal firing rates of 5 to 50 Hz and intermittent rates up to 250 Hz, leaving a substantial fraction of synaptic receptors occupied persistently by GABA. Persistent exposure of neurons to GABA has been shown to cause a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine (BZD) site interactions with a half-life of approximately 24 h. Here, we report that a single brief exposure of neocortical neurons in primary culture to GABA for 5-10 min (t(1/2) = 3.2 +/- 0.2 min) initiates a process that results in uncoupling hours later (t(1/2) = 12.1 +/- 2.2 h). Initiation of delayed-onset uncoupling is blocked by co-incubation with picrotoxin or alpha-amanitin but is insensitive to nifedipine, indicating that uncoupling is contingent upon receptor activation and transcription but is not dependent on voltage-gated Ca2+ influx. Delayed-onset uncoupling occurs without a change in receptor number or a change in the proportion of alpha1 subunit pharmacology, as zolpidem binding affinity is unaltered. Such activity dependent latent modulation of GABA(A)R function that manifests as delayed-onset uncoupling may be relevant to physiological, pathophysiological, and pharmacological conditions where synaptic receptors are transiently exposed to GABA agonists for several minutes.