Martin Mortensen

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.

GABA potency at GABA A receptors found in synaptic and extrasynaptic zones

The potency of GABA is vitally important for its primary role in activating GABAA receptors and acting as an inhibitory neurotransmitter. Although numerous laboratories have presented information, directly or indirectly, on GABA potency, it is often difficult to compare across such studies given the inevitable variations in the methods used, the cell types studied, whether native or recombinant receptors are examined, and their relevance to native synaptic and extrasynaptic GABAA receptors. In this review, we list the most relevant isoforms of synaptic and extrasynaptic GABAA receptors that are thought to assemble in surface membranes of neurons in the central nervous system. Using consistent methodology in one cell type, the potencies of the endogenous neurotransmitter GABA are compared across a spectrum of GABAA receptors. The highest potency for GABA is measured when activating extrasynaptic-type α6 subunit-containing receptors, whereas synaptic-type α2β3γ2 and α3β3γ2 receptors exhibited the lowest potency, and other GABAA receptor subtypes that are found both in synaptic and extrasynaptic compartments, showed intermediate sensitivities to GABA. The relatively simple potency relationship between GABA and its target receptors is important as it serves as one of the major determinants of GABAA receptor activation, with consequences for the development of inhibition, either by tonic or phasic mechanisms.

Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptors

The activation characteristics of synaptic and extrasynaptic GABAA receptors are important for shaping the profile of phasic and tonic inhibition in the central nervous system, which will critically impact on the activity of neuronal networks. Here, we study in isolation the activity of three agonists, GABA, muscimol and 4,5,6,7‐tetrahydoisoxazolo[5,4‐c]pyridin‐3(2H)‐one (THIP), to further understand the activation profiles of α1β3γ2, α4β3γ2 and α4β3δ receptors that typify synaptic‐ and extrasynaptic‐type receptors expressed in the hippocampus and thalamus. The agonists display an order of potency that is invariant between the three receptors, which is reliant mostly on the agonist dissociation constant. At δ subunit‐containing extrasynaptic‐type GABAA receptors, both THIP and muscimol additionally exhibited, to different degrees, superagonist behaviour. By comparing whole‐cell and single channel currents induced by the agonists, we provide a molecular explanation for their different activation profiles. For THIP at high concentrations, the unusual superagonist behaviour on α4β3δ receptors is a consequence of its ability to increase the duration of longer channel openings and their frequency, resulting in longer burst durations. By contrast, for muscimol, moderate superagonist behaviour was caused by reduced desensitisation of the extrasynaptic‐type receptors. The ability to specifically increase the efficacy of receptor activation, by selected exogenous agonists over that obtained with the natural transmitter, may prove to be of therapeutic benefit under circumstances when synaptic inhibition is compromised or dysfunctional.

Extrasynaptic αβ subunit GABAA receptors on rat hippocampal pyramidal neurons

Extrasynaptic GABAA receptors that are tonically activated by ambient GABA are important for controlling neuronal excitability. In hippocampal pyramidal neurons, the subunit composition of these extrasynaptic receptors may include α5βγ and/or α4βδ subunits. Our present studies reveal that a component of the tonic current in the hippocampus is highly sensitive to inhibition by Zn2+. This component is probably not mediated by either α5βγ or α4βδ receptors, but might be explained by the presence of αβ isoforms. Using patch‐clamp recording from pyramidal neurons, a small tonic current measured in the absence of exogenous GABA exhibited both high and low sensitivity to Zn2+ inhibition (IC50 values, 1.89 and 223 μm, respectively). Using low nanomolar and micromolar GABA concentrations to replicate tonic currents, we identified two components that are mediated by benzodiazepine‐sensitive and ‐insensitive receptors. The latter indicated that extrasynaptic GABAA receptors exist that are devoid of γ2 subunits. To distinguish whether the benzodiazepine‐insensitive receptors were αβ or αβδ isoforms, we used single‐channel recording. Expressing recombinant α1β3γ2, α5β3γ2, α4β3δ and α1β3 receptors in human embryonic kidney (HEK) or mouse fibroblast (Ltk) cells, revealed similar openings with high main conductances (∼25–28 pS) for γ2 or δ subunit‐containing receptors whereas αβ receptors were characterized by a lower main conductance state (∼11 pS). Recording from pyramidal cell somata revealed a similar range of channel conductances, indicative of a mixture of GABAA receptors in the extrasynaptic membrane. The lowest conductance state (∼11 pS) was the most sensitive to Zn2+ inhibition in accord with the presence of αβ receptors. This receptor type is estimated to account for up to 10% of all extrasynaptic GABAA receptors on hippocampal pyramidal neurons.

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), isonipecotic acid, piperidine‐4‐sulphonic acid (P4S), imidazole‐4‐acetic acid (IAA), 5‐(4‐piperidyl)‐3‐isothiazolol (thio‐4‐PIOL) and 5‐(4‐piperidyl)‐3‐isoxazolol (4‐PIOL). Whole‐cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5‐fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch‐like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

Single-channel recording of ligand-gated ion channels

Electrophysiological recording of single-channel currents is the most direct method available for obtaining detailed and precise information about the kinetic behavior of ion channels. A wide variety of cell types can be used for single-channel recording, but to obtain the highest resolution of the briefest channel opening and closing events, low-noise recordings, coupled with a minimal filtering frequency, are required. Here, we present a protocol designed to help those with some electrophysiological expertise who wish to explore the properties of native and recombinant single ligand-gated ion channels. We have focused on the practical aspects of recording single GABA channels from cell-attached and outside-out patches and also introduced some of the preliminary considerations that are necessary for the analysis of single-channel data, including an introduction to single-channel analysis software.

Characterization of GABAA receptor ligands in the rat cortical wedge preparation: evidence for action at extrasynaptic receptors?

GABAA receptor agonists have previously been characterized at human GABAA receptors expressed in Xenopus oocytes. The correlation between these data and functional in vivo data of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP) has shown that THIP is 100 fold more potent in clinical studies than in oocytes. THIP and a series of agonists (GABA, Isoguvacine), partial agonists (Imidazole acetic acid; P4S, 4‐PIOL, thio‐4‐PIOL) and one antagonist (SR95531) were characterized in the rat cortical wedge preparation using inhibition of spontaneous activity in Mg++ free medium as the measurable parameter. Agonists were in general 40 times more potent in the wedge preparation than at α1β3γ2s containing receptors expressed in Xenopus oocytes, whereas the antagonist was equipotent under these two conditions. Partial agonists with responses above 6% at α1β3γ2s containing receptors were full agonists in the rat cortical wedge preparation, whereas partial agonists with maximum responses below 6% behaved as partial agonists in the rat cortical wedge preparation. These data suggest that only a small fraction of the GABAA receptors in the rat cortical wedge needs to be activated by GABAA agonists in order to obtain a maximum response. Results therefore indicate a significant contribution of extrasynaptic receptors to pharmacological activity of exogenous applied GABAA agonists in this system.

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.

Bioisosteric determinants for subtype selectivity of ligands for heteromeric GABAA receptors

The potency and efficacy of a series of bioisosterically modified GABA analogues were determined electrophysiologically using heteromeric GABA(A) receptors expressed in Xenopus oocytes. These agonist parameters were shown to be strongly dependent on the receptor subunit combination. On the other hand, the antagonist potencies of the classical GABA(A) antagonists SR 95531 (7) and BMC (8) and also of 5g and the phosphinic acid bioisosteres of 5a, compounds 5f and 6, were essentially independent of the receptor subunit combinations.

Stoichiometry of δ subunit containing GABAA receptors

Although the stoichiometry of the major synaptic αβγ subunit‐containing GABAA receptors has consensus support for 2α:2β:1γ, a clear view of the stoichiometry of extrasynaptic receptors containing δ subunits has remained elusive. Here we examine the subunit stoichiometry of recombinant α4β3δ receptors using a reporter mutation and a functional electrophysiological approach.