Effect of Ethanol Treatment on Rate and Equilibrium Constants for [³H]Muscimol Binding to Rat Brain Membranes: Alteration of Two Affinity States of the GABA_A Receptor

Abstract

Equilibrium binding curves were biphasic in control and ethanol‐treated rats. [3H]Muscimol binds to sites of high (KDA of ∼10 nM) and low (KDB of ∼0.3–0.4 µM) affinity. Chronic ethanol treatment produced a decrease in BmaxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high‐affinity site. IC50 and Ki values were calculated for several competing ligands, using membranes from both control and ethanol‐treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low‐affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol‐treated animals for both phases.

Extrasynaptic δ‐GABAA receptors are high‐affinity muscimol receptors

“…4d). The binding affinities, number of [ 3 H]muscimol binding sites as well as binding kinetics are in the same range as found in the literature (Wang et al 1979;Agey and Dunn 1989;Maksay 1990;Negro et al 1995;Ebert et al 1999). However, because of the missing high-affinity d-receptors with slow kinetics, our association and dissociation rates in dKO fore/midbrain, dKO cerebellum, and recombinant a1b2c2 receptors are an exception as they were faster than all association rates in the former published studies.…”

“…In addition, chronic ethanol treatment leads to a substantial reduction in high‐affinity [ 3 H]muscimol‐binding sites (Negro et al . ), which meshes well with the notion that chronic alcohol leads to a reduction in δ‐GABA A R‐mediated tonic currents and δ‐subunit surface expression, a process that likely contributes to alcohol tolerance and the development of alcohol dependence (for review see Olsen and Liang ).…”

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABAA receptors after chronic ethanol exposure

“…The decreased number of [ 3 H]muscimol binding sites in cerebral cortex observed after chronic ingestion in wild‐type mice is absent in knockout mice, suggesting that, as observed for NMDA receptors, the neuroadaptations of GABA A receptors following chronic ethanol are blunted in mice lacking CB1 receptors. A decreased number of high‐affinity sites for [ 3 H]muscimol has already been demonstrated in rat brain cortex after chronic ethanol treatment (Negro et al. 1995).…”

GABAergic and glutamatergic effects on nigrostriatal and mesolimbic dopamine release in the rat

“…Aim MUS is as highly selective GABA A R agonist (inhibition constant [K i ] = 2.7 nM; Negro et al 1995). We had previously shown that D 2 R-like imaging can be employed in order to estimate alterations of synaptic DA upon challenge with compounds such as methylphenidate and L-DOPA, which modify DA availability in the synaptic cleft (for review see Nikolaus et al 2011).…”