Alastair M. Hosie

Endogenous neurosteroids regulate GABA A receptors through two discrete transmembrane sites

Inhibitory neurotransmission mediated by GABAA 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 GABAA 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 α-subunit transmembrane domains, whereas direct receptor activation is initiated by interfacial residues between α and β 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 GABAA 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.

Zinc-mediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity.

Zinc ions are concentrated in the central nervous system and regulate GABAA receptors, which are pivotal mediators of inhibitory synaptic neurotransmission. Zinc ions inhibit GABAA receptor function by an allosteric mechanism that is critically dependent on the receptor subunit composition: αβ subunit combinations show the highest sensitivity, and αβγ isoforms are the least sensitive. Here we propose a mechanistic and structural basis for this inhibition and its dependence on the receptor subunit composition. We used molecular modeling to identify three discrete sites that mediate Zn2+ inhibition. One is located within the ion channel, and the other two are on the external amino (N)-terminal face of the receptor at the interfaces between α and β subunits. We found that the characteristically low Zn2+ sensitivity of GABAA receptors containing the γ2 subunit results from disruption to two of the three sites after receptor subunit co-assembly.

Zn2+ Ions: Modulators of Excitatory and Inhibitory Synaptic Activity

The role of Zn2+ in the CNS has remained enigmatic for several decades. This divalent cation is accumulated by specific neurons into synaptic vesicles and can be released by stimulation in a Ca2+-dependent manner. Using Zn2+ fluorophores, radiolabeled Zn2+, and selective chelators, the location of this ion and its release pattern have been established across the brain. Given the distribution and possible release under physiological conditions, Zn2+ has the potential to act as a modulator of both excitatory and inhibitory neurotransmission. Excitatory N-methyl-D-aspartate (NMDA) receptors are directly inhibited by Zn2+, whereas non-NMDA receptors appear relatively unaffected. In contrast, inhibitory transmission mediated via GABAAreceptors can be potentiated via a presynaptic mechanism, influencing transmitter release; however, although some tonic GABAergic inhibition may be suppressed by Zn2+, most synaptic GABA receptors are unlikely to be modulated directly by this cation. In the spinal cord, glycinergic transmission may also be affected by Zn2+ causing potentiation. Recently, the penetration of synaptically released Zn2+ into neurons suggests that this ion has the potential to act as a direct transmitter, by affecting postsynaptic signaling pathways. Taken overall, present studies are broadly supportive of a neuromodulatory role for Zn2+ at specific excitatory and inhibitory synapses.

Dynamic mobility of functional GABAA receptors at inhibitory synapses

Importing functional GABAA receptors into synapses is fundamental for establishing and maintaining inhibitory transmission and for controlling neuronal excitability. By introducing a binding site for an irreversible inhibitor into the GABAA receptor α1 subunit channel lining region that can be accessed only when the receptor is activated, we have determined the dynamics of receptor mobility between synaptic and extrasynaptic locations in hippocampal pyramidal neurons. We demonstrate that the cell surface GABAA receptor population shows no fast recovery after irreversible inhibition. In contrast, after selective inhibition, the synaptic receptor population rapidly recovers by the import of new functional entities within minutes. The trafficking pathways that promote rapid importation of synaptic receptors do not involve insertion from intracellular pools, but reflect receptor diffusion within the plane of the membrane. This process offers the synapse a rapid mechanism to replenish functional GABAA receptors at inhibitory synapses and a means to control synaptic efficacy.

Conserved site for neurosteroid modulation of GABAA receptors

This study addresses whether the potentiation site for neurosteroids on GABA(A) receptors is conserved amongst different GABA(A) receptor isoforms. The neurosteroid potentiation site was previously identified in the alpha1beta2gamma2S receptor by mutation of Q241 to methionine or leucine, which reduced the potentiation of GABA currents by the naturally occurring neurosteroids, allopregnanolone or tetrahydrodeoxycorticosterone (THDOC). By using heterologous expression of GABA(A) receptors in HEK cells, in combination with whole-cell patch clamp recording methods, a relatively consistent potentiation by allopregnanolone of GABA-activated currents was evident for receptors composed of one alpha subunit isoform (alpha2-5) assembled with beta3 and gamma2S subunits. Using mutant alphabetagamma receptors, the neurosteroid potentiation was universally dependent on the conserved glutamine residue in M1 of the respective alpha subunit. Studying wild-type and mutant receptors composed of alpha4beta3delta subunits revealed that the delta subunit is unlikely to contribute to the neurosteroid potentiation binding site and probably affects the efficacy of potentiation. Thus, in keeping with the ability of neurosteroids to potentiate GABA currents via a broad variety of GABA(A) receptor isoforms in neurons, the potentiation site is structurally highly conserved on this important neurotransmitter receptor family.

Mutations in the Gabrb1 gene promote alcohol consumption through increased tonic inhibition

Alcohol-dependence is a common, complex and debilitating disorder with genetic and environmental influences. Here we show that alcohol consumption increases following mutations to the γ-aminobutyric acidA receptor (GABAAR) β1 subunit gene (Gabrb1). Using N-ethyl-N-nitrosourea mutagenesis on an alcohol-averse background (F1 BALB/cAnN × C3H/HeH), we develop a mouse model exhibiting strong heritable preference for ethanol resulting from a dominant mutation (L285R) in Gabrb1. The mutation causes spontaneous GABA ion channel opening and increases GABA sensitivity of recombinant GABAARs, coupled to increased tonic currents in the nucleus accumbens, a region long-associated with alcohol reward. Mutant mice work harder to obtain ethanol, and are more sensitive to alcohol intoxication. Another spontaneous mutation (P228H) in Gabrb1 also causes high ethanol consumption accompanied by spontaneous GABA ion channel opening and increased accumbal tonic current. Our results provide a new and important link between GABAAR function and increased alcohol consumption that could underlie some forms of alcohol abuse.

Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

Regulation of GABAA receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABAA receptors at alkaline pH has remained elusive. We report here that the GABA‐activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit‐containing receptors, but only at GABA concentrations below the EC40. Site‐specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABAA receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2H267, the mutation γ2T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABAA receptor.

Replacement of asparagine with arginine at the extracellular end of the second transmembrane (M2) region of insect GABA receptors increases sensitivity to penicillin G

The actions of penicillin-G (PCG) on wild-type and mutant Drosophila GABA receptor (RDL) subunits expressed in Xenopus oocytes were studied under two-electrode voltage-clamp. PCG was found to be a non-competitive antagonist of homomeric Drosophila RDL receptors with an IC50 of 20.41xa0±xa01.66xa0mM at EC50 GABA. Substitution of a single amino acid (N318R) at the extracellular end of the channel lining region of the RDL subunit increased the potency of GABA approximately four fold, and increased the IC50 of PCG to 5.09xa0±xa00.38xa0mM. Although the antagonism by PCG on wild-type RDL receptors was independent of membrane potential, PCG action on the N318R mutant showed pronounced voltage-dependency, being much more effective at positive membrane potentials. Thus, in RDL homomers, the replacement of N318 by R318, a residue present at the equivalent position in vertebrate GABAA receptors, confers a vertebrate-like PCG pharmacology to the N318R mutant receptor. The A301S mutation that confers resistance to dieldrin did not significantly affect the antagonism by PCG.