Inhibitory neurotransmission mediated by GABA(A) receptors can be modulated by the endogenous neurosteroids, allopregnanolone and tetrahydro-deoxycorticosterone. Neurosteroids are synthesized de novo in the brain during stress, pregnancyand after ethanol consumption, and disrupted steroid regulation of GABAergic transmission is strongly implicated in several debilitating conditions such as panic disorder, major depression, schizophrenia, alcohol dependence and catamenial epilepsy. Determining how neurosteroids interact with the GABA(A) receptor is a prerequisite for understanding their physiological and pathophysiological roles in the brain. Here we identify two discrete binding sites in the receptor's transmembrane domains that mediate the potentiating and direct activation effects of neurosteroids. They potentiate GABA responses from a cavity formed by the alpha-subunit transmembrane domains, whereas direct receptor activation is initiated by interfacial residues between alpha and beta subunits and is enhanced by steroid binding to the potentiation site. Thus, significant receptor activation by neurosteroids relies on occupancy of both the activation and potentiation sites. These sites are highly conserved throughout the GABA(A )receptor family, and their identification provides a unique opportunity for the development of new therapeutic, neurosteroid-based ligands and transgenic disease models of neurosteroid dysfunction.

The divalent cation Zn 2؉ is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn 2؉ . Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn 2؉ potentiation site on GlyRs and account for the differential sensitivity of GlyR ␣ 1 and GlyR ␣ 2 to Zn 2؉ potentiation. In addition, juxtaposed to this Zn 2؉ site, which is located externally on the N-terminal domain of the ␣ subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn 2؉ potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn 2؉ binds to a site on the extracellular outer face of the GlyR ␣ subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.

1 Extracellular single unit recording techniques were used to compare the e ects of (-)-3-amino-1-hydroxypyrrolidin-2-one ((7)-HA-966) and (+)-baclofen on the activity of dopamine-containing neurones in 300 mm slices of rat substantia nigra. Electrophysiological data were compared with the outcome of in vitro binding experiments designed to assess the a nity of (7)-HA-966 for g-aminobutyric acid (GABA B ) receptors. 2 Bath application of (7)-HA-966 produced a concentration-dependent inhibition of dopaminergic neuronal ®ring (EC 50 =444.0 mM; 95% con®dence interval: 277.6 mM ± 710.1 mM, n=27) which was fully reversible upon washout from the recording chamber. Although similar e ects were observed in response to (+)-baclofen, the direct-acting GABA B receptor agonist proved to be considerably more potent than (7)-HA-966 (EC 50 =0.54 mM; 95% con®dence interval: 0.44 mM ± 0.66 mM, n=29) in vitro. 3 Low concentrations of chloral hydrate (10 mM) were without e ect on the basal ®ring rate of nigral dopaminergic neurones but signi®cantly increased the inhibitory e ects produced by concomitant application of (7)-HA-966. 4 The inhibitory e ects of (7)-HA-966 were completely reversed in the presence of the GABA B receptor antagonists, CGP-35348 (100 mM) and 2-hydroxysaclofen (500 mM). Bath application of CGP-35348 alone increased basal ®ring rate. However, the magnitude of the excitation (9.2+0.3%) was not su cient to account for the ability of the antagonist to reverse fully the inhibitory e ects of (7)-HA-966. 5 (7)-HA-966 (0.1 ± 1.0 mM) produced a concentration-dependent displacement of [ 3 H]-GABA from synaptic membranes in the presence of isoguvacine (40 mM). However, the a nity of the drug for GABA B binding sites was signi®cantly less than that of GABA (0.0005 potency ratio) and showed no apparent stereoselectivity. 6 These results indicate that while (7)-HA-966 appears to act as a direct GABA B receptor agonist in vitro, its a nity for this receptor site is substantially less than that of GABA or baclofen and unlikely to account for the depressant actions of this drug which occur at levels approximately ten fold lower in vivo.