Trevor G. Smart

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.

Cloning and functional expression of a brain G-protein-coupled ATP receptor

A cDNA encoding a novel member of the G‐protein‐coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly‐developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.

GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization

Prostaglandin E2 (PGE2) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PGE2-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy.

Constructing inhibitory synapses

Control of nerve-cell excitability is crucial for normal brain function. Two main groups of inhibitory neurotransmitter receptors — GABAA and glycine receptors — fulfil a significant part of this role. To mediate fast synaptic inhibition effectively, these receptors need to be localized and affixed opposite nerve terminals that release the appropriate neurotransmitter at multiple sites on postsynaptic neurons. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of mature, functional inhibitory synaptic receptor proteins. This review describes the dynamic regulation of synaptic GABAA and glycine receptors and discusses recent advances in this rapidly evolving field.

Regulation of GABAA receptor function by protein kinase C phosphorylation.

GABAA receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of beta and gamma 2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in beta 1 and gamma 2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the gamma 2 subunit exerted the largest effect on the GABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.

Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability.

The efficacy of GABAergic synaptic inhibition is a principal factor in controlling neuronal activity. We demonstrate here that brain-derived neurotrophic factor modulates the activity of GABAA receptors, the main sites of fast synaptic inhibition in the brain, within minutes of application. Temporally, this comprised an early enhancement in the miniature IPSC amplitude, followed by a prolonged depression. This modulation was concurrent with enhanced PKC-mediated phosphorylation, followed by protein phosphatase 2A (PP2A)-mediated dephosphorylation of the GABAA receptor. Mechanistically, these events were facilitated by differential recruitment of PKC, receptor for activated C-kinase, and PP2A to GABAA receptors, depending on the phosphorylation state of the receptor β3-subunit. Thus, transient formation of GABAA receptor signaling complexes has the potential to provide a basis for acute changes in receptor function underlying GABAergic synaptic plasticity.

Retrograde activation of presynaptic NMDA receptors enhances GABA release at cerebellar interneuron–Purkinje cell synapses

Synaptic inhibition is a vital component in the control of cell excitability within the brain. Here we report a newly identified form of inhibitory synaptic plasticity, termed depolarization-induced potentiation of inhibition, in rodents. This mechanism strongly potentiated synaptic transmission from interneurons to Purkinje cells after the termination of depolarization-induced suppression of inhibition. It was triggered by an elevation of Ca2+ in Purkinje cells and the subsequent retrograde activation of presynaptic NMDA receptors. These glutamate receptors promoted the spontaneous release of Ca2+ from presynaptic ryanodine-sensitive Ca2+ stores. Thus, NMDA receptor–mediated facilitation of transmission at this synapse provides a regulatory mechanism that can dynamically alter the synaptic efficacy at inhibitory synapses.

GABA A receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1

Controlling the number of functional γ-aminobutyric acid A (GABAA) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABAA receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABAA receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABAA receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABAA receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.

GABAA receptors are differentially sensitive to zinc: dependence on subunit composition.

GABAA receptors with different subunit composition, were expressed in kidney cells and studied by whole cell recording. Expressed GABAA receptors were differentially sensitive to inhibition by zinc; receptors which lacked the γ subunit were inhibited by zinc. Embryonic neurones also exhibited zinc‐sensitive GABA responses, in contrast to adult neurones. This developmentally‐sensitive aspect of GABAA receptor pharmacology may be partly dependent on expression of the γ subunit.