Recombinant Alpha1beta2gamma2 Receptors Research Articles

Antagonism of neurosteroid modulation of native γ-aminobutyric acid receptors by (3α,5α)-17-phenylandrost-16-en-3-ol

Endogenous pregnane neurosteroids are allosteric modulators at gamma-aminobutyric acid type-A (GABAA) receptors at nanomolar concentrations. There is direct evidence for multiple distinct neurosteroid binding sites on GABAA receptors, dependent upon subunit composition and stoichiometry. This view is supported by the biphasic kinetics of various neuroactive steroids, enantioselectivity of some neurosteroids, selective mutation studies of recombinantly expressed receptors and the selectivity of the neurosteroid antagonist (3alpha,5alpha)-17-phenylandrost-16-en-3-ol (17PA) on 5alpha-pregnane steroid effects on recombinant GABAA receptors expressed in Xenopus oocytes and native receptors in dissociated neurons. However, it is unclear whether this antagonist action is present in a mature mammalian system. The present study evaluated the antagonist activity of 17PA on neurosteroid agonists both in vivo and in vitro by examining the effects of 17PA on 5alpha-pregnane-induced sedation in rats, native mature GABAA receptor ion channels utilizing the chloride flux assay and further studies in recombinant alpha1beta2gamma2 receptors. The data show that 17PA preferentially inhibits 3alpha,5alpha-THP vs. alphaxalone in vivo, preferentially inhibits 3alpha,5alpha-THDOC vs. alphaxalone potentiation of GABA-mediated Cl- uptake in adult cerebral cortical synaptoneurosomes, but shows no specificity for 3alpha,5alpha-THDOC vs. alphaxalone in recombinant alpha1beta2gamma2 receptors. These data provide further evidence of the specificity of 17PA and the heterogeneity of neurosteroid recognition sites on GABAA receptors in the CNS.

Evidence for a Reduction of Coupling between GABAA Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[35S]TBPS binding to the GABAA receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [35S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [35S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [3H]flunitrazepam binding to benzodiazepine site of recombinant alpha1beta2gamma2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [35S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABAA receptors.

A gain‐of‐function mutation in the GABAA receptor produces synaptic and behavioral abnormalities in the mouse

In mammalian species, inhibition in the brain is mediated predominantly by the activation of GABAA receptors. We report here changes in inhibitory synaptic function and behavior in a mouse line harboring a gain-of-function mutation at Serine 270 (S270) in the GABAA receptor alpha1 subunit. In recombinant alpha1beta2gamma2 receptors, replacement of S270 by Histidine (H) results in an increase in sensitivity to gamma-aminobutyric acid (GABA), and slowing of deactivation following transient activation by saturating concentrations of GABA. Heterozygous mice expressing the S270H mutation are hyper-responsive to human contact, exhibit intention tremor, smaller body size and reduced viability. These mice also displayed reduced motor coordination, were hypoactive in the home cage, but paradoxically were hyperactive in a novel open field environment. Heterozygous knockin mice of both sexes were fertile but females failed to care for offspring. This deficit in maternal behavior prevented production of homozygous animals. Recordings from brain slices prepared from these animals revealed a substantial prolongation of miniature inhibitory postsynaptic currents (IPSCs) and a loss of sensitivity to the anesthetic isoflurane, in neurons that express a substantial amount of the alpha1 subunit. The results suggest that the biophysical properties of GABAA receptors are important in determining the time-course of inhibition in vivo, and suggest that the duration of synaptic inhibition is a critical determinant that influences a variety of behaviors in the mouse.

Enantioselectivity of alpha-benzyl-alpha-methyl-gamma-butyrolactone-mediated modulation of anticonvulsant activity and GABA(A) receptor function.

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA(A) receptors. Lactones with small alkyl substitutions at the alpha-position positively modulate the channel, whereas beta-substituted lactones tend to inhibit the GABA(A) receptor. These compounds mediate inhibition through the picrotoxin site of the receptor. A distinct binding site that mediates the stimulatory actions of lactones is presumed to exist, although no definitive evidence to support this claim exists. In the present study, we used in vivo and in vitro assays to evaluate the effects of the enantiomers of a novel lactone, alpha-benzyl-alpha-methyl-gamma-butyrolactone (alpha-BnMeGBL), on the GABA(A) receptor. R-(-)-alpha-BnMeGBL was 2-fold more potent than the S-(+)-alpha-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC(50) of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant alpha1beta2gamma2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-alpha-BnMeGBL than that seen with (-)-alpha-BnMeGBL. Both enantiomers of alpha-BnMeGBL directly gated the GABA(A) receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of alpha-BnMeGBL on GABA(A) receptor function display enantioselectivity and provide strong evidence for the existence of a true "lactone site" on the receptor.

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.