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Pressure-sensitive and -insensitive coupling in gamma-aminobutyric acid(A) receptors
D L Davies1, L D McCauley, M B Bolger
1Alcohol and Brain Research Laboratory, Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA. ddavies@hsc.usc.edu
Rationale:
Previous behavioral and biochemical studies suggest that allosteric coupling processes initiated by benzodiazepines, barbiturates and neuroactive steroids can be sub-categorized on the basis of their sensitivities to antagonism by increased atmospheric pressure. However, biochemical evidence supporting this hypothesis was limited to single concentration studies in long sleep (LS) mice.
Objective:
The present paper addresses these issues by extending biochemical investigation of pressure effects on allosteric modulators across a range of concentrations that allosterically enhance gamma-aminobutyric acid (GABA)A receptor function and alter behavior using two mouse genotypes. In addition, the effects of pressure on ligand binding were explored to further investigate the mechanism of pressure antagonism of allosteric modulation.
Methods:
The effects of 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) on allosteric modulation of GABA(A) receptor function and [3H]flunitrazepam binding was tested in LS and C57BL mouse brain membranes (microsacs) using chloride flux and high-affinity binding assays.
Results:
In both genotypes, exposure to 12 ATA heliox antagonized the allosteric enhancement of GABA(A) receptor function by flunitrazepam (0.1-10 microM) and pentobarbital (0.1-50 microM) but did not affect allosteric modulation by 3alpha-hydroxy-5beta-pregnan-20-one (0.1-1 microM). Pressure did not affect benzodiazepine receptor affinity (Kd) or the number of benzodiazepine receptors (Bmax).
The Results:
(1) confirm that there are differences in sensitivity to pressure antagonism of allosteric coupling among GABA(A) allosteric modulators; (2) demonstrate that these differences are not concentration or genotype dependent; (3) add evidence that pressure antagonizes allosteric modulation by uncoupling the receptor and (4) support the hypothesis that allosteric modulation of receptor function can be sub-categorized on the basis of sensitivity to pressure antagonism.