Benzodiazepine agonist and inverse agonist coupling in GABA_A receptors antagonized by increased atmospheric pressure

Abstract

Past work found that exposure to 12 times normal atmospheric pressure (ATA) of helium–oxygen gas (heliox) selectively antagonizes (uncouples) and differentiates allosteric coupling in GABA_A receptors initiated by benzodiazepines versus neurosteroids. The present study tested the hypothesis that pressure can differentiate coupling initiated by a spectrum of benzodiazepine receptor ligands by measuring the effects of pressure on benzodiazepine ligand modulation of GABA-activated ³⁶Cl⁻ uptake in mouse brain membranes. 12 ATA completely antagonized allosteric modulation by: benzodiazepine receptor agonists diazepam and flunitrazepam; Type-1 selective benzodiazepine receptor agonist zolpidem and the benzodiazepine receptor partial inverse agonist ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-α][1,4]benzodiazepine-3-carboxylate (Ro15-4513). The similar, non-competitive-like characteristics of pressure antagonism of these ligands suggest common structural/functional elements underlying their coupling. Pressure also antagonized allosteric modulation by the benzodiazepine receptor inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), but the antagonism was not complete and appeared to be surmountable (competitive-like) suggesting unexpected differences in coupling for DMCM versus Ro15-4513. These studies represent the first attempt to use pressure as a tool to dissect benzodiazepine receptor coupling. The results suggest that there is a common, pressure antagonism sensitive structural/functional element underlying coupling for benzodiazepine receptor ligands and that coupling for the full inverse benzodiazepine receptor agonist DMCM differs from coupling for benzodiazepine receptor agonists and benzodiazepine receptor partial inverse agonists.

Introduction

GABA_A receptors are allosterically modulated by several classes of compounds acting at distinct recognition binding sites including sites for benzodiazepines, barbiturates and neuroactive steroids Macdonald and Olsen, 1994, McCauley et al., 1994, Sieghart, 1995, Smith and Olsen, 1995. Allosteric modulation involves three distinct events: (1) ligand binding to the benzodiazepine recognition site, (2) transduction of the signal (coupling) to the GABA effector site and (3) alteration in GABA-gated currents Macdonald and Olsen, 1994, McCauley et al., 1994, Smith and Olsen, 1995, Davies et al., 2001.

The benzodiazepine binding region of GABA_A receptors is pharmacologically unique in that benzodiazepine receptor ligands can be arranged on a continuum according to their efficacy ranging from full agonists to full inverse agonist Smith and Olsen, 1995, Lüddens and Korpi, 1996. Positive benzodiazepine receptor ligands (e.g., diazepam) induce their effects by increasing frequency of Cl⁻ channel opening without a change in the single-channel conductance, whereas negative (inverse) agonist benzodiazepine receptor ligands (e.g., methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate [DMCM]) induce their effects by decreasing channel open frequency Rogers et al., 1994, Bianchi and Macdonald, 2001. Benzodiazepine ligands do not alter Cl⁻ channels in the absence of GABA Rogers et al., 1994, Smith and Olsen, 1995.

The molecular architecture and function of benzodiazepine ligand binding regions on GABA_A receptors have been extensively investigated, but differences in the benzodiazepine binding site do not appear to explain differences in the functional effects of benzodiazepine receptor ligands Olsen and Tobin, 1990, Pritchett and Seeburg, 1991, Macdonald and Olsen, 1994, Smith and Olsen, 1995, Sieghart, 1995, Kelly et al., 2002. Only recently have studies begun to focus on the structural and functional bases mediating allosteric coupling induced by ligands binding to the benzodiazepine receptor site. Overall, these studies, using molecular manipulations, suggest that functional differences between the effects of benzodiazepine receptor agonists and inverse agonists may reflect subtle differences in the structural determinants underlying coupling for respective ligands Mihic et al., 1994, Buhr et al., 1996, Boileau et al., 1998, Boileau and Czajkowski, 1999, Carlson et al., 2000, Williams and Akabas, 2000, Kelly et al., 2002.

Despite these advances, progress in understanding the role coupling plays in mediating functional differences in benzodiazepine ligands is slowed by the difficulties in manipulating coupling directly, since available pharmacological tools act via interference with binding (e.g., flumazenil), by blocking the GABA effector (e.g., bicuculline) or by blocking the channel (e.g., picrotoxin). Hence, pharmacological agents do not act directly on coupling and cannot be used in the classical manner to tease apart differences in mechanisms and structures mediating coupling.

Recent findings suggest that increased atmospheric pressure is a direct, selective antagonist of allosteric coupling that can help fill this void. These studies found that exposure to 12 times normal atmospheric pressure (12 ATA) of helium–oxygen gas (heliox) antagonized flunitrazepam and pentobarbital potentiation of GABA-activated Cl⁻ uptake, but did not antagonize potentiation by the neuroactive steroid, 3α-hydroxy-5β-pregnan-20-one Davies et al., 1999, Davies et al., 2001. These biochemical studies also found that exposure to 12 ATA heliox did not affect benzoodiazepine receptor affinity (K_d) or the number of benzodiazepine receptors (B_max), thus providing evidence that pressure does not antagonize allosteric modulators by altering binding (Davies et al., 2001). Behavioral studies found similar selectivity of pressure Davies et al., 1996, Davies et al., 1999. Exposure to 12 ATA heliox did not alter the effects of GABA on GABA_A receptor function in the absence of allosteric modulators Davies and Alkana, 1998, Davies et al., 1999. Collectively, these findings indicate that: (1) Pressure directly antagonizes BZ-induced allosteric modulation by uncoupling the benzodiazepine receptor binding site from the GABA effector (Davies et al., 2001) and (2) allosteric modulation of GABA_A receptor function can be sub-categorized on the basis of sensitivity to pressure antagonism (Davies et al., 2001).

The present study begins to test the hypothesis that pressure can differentiate coupling initiated by a spectrum of benzodiazepine receptor ligands with different functional properties. This was accomplished by testing the effects of 12 ATA heliox versus coupling initiated by: (1) non-selective benzodiazepine receptor agonists (diazepam and flunitrazepam); (2) imidazopyridine Type-1 selective benzodiazepine receptor agonist (zolpidem); (3) imidazobenzodiazepine benzodiazepine receptor partial inverse agonist (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-α][1,4]benzodiazepine-3-carboxylate [Ro15-4513]) and 4) β-carboline full benzodiazepine receptor inverse agonist (DMCM). The results suggest that there is a common, pressure antagonism sensitive structural/functional element underlying coupling for benzodiazepine agonist and inverse agonist ligands. Differences in characteristics of the antagonism between DMCM and the other benzodiazepine receptor ligands tested suggest that there may be subtle differences in coupling of benzodiazepine receptor full inverse agonists versus coupling for benzodiazepine receptor partial inverse agonists and agonists.