Our research

Molecular mechanisms of ion channel function
Research overview:
The ligand-gated ion channels combine the functionalities of a receptor and an ion channel in a single protein. g-aminobutyric acid (GABA) gates the Cl- selective channel of GABA_A receptors that function by mediating both a fast synaptic signalling and a basal tonic inhibition in the central nervous system. I endeavour to understand the function of these multi-subunit protein complexes from the perspective of a protein chemist, in both native and model systems. Recombinant expression systems are used for both the functional analysis of intact receptors in host cells and also structural studies of soluble domains. The study of native receptors in neurons involves the use of electrophysiology, confocal microscopy, qRT-PCR and siRNA gene expression knockdown. The study of model receptors includes the use of electrophysiology, molecular biology, biochemistry, confocal and electron microscopy and X-ray crystallography. The functional studies are focused on GABA_A receptors with an emphasis on drug modulation and receptor clustering, while structural studies include additional members of this protein family.

Current Projects

The physical mechanism and functional consequences of GABA_A receptor clustering
High conductance single-channels (>40 pS) have been reported in studies on GABA_A receptors in situ but recombinant GABA_A channels expressed in heterologous expression systems do not display such high conductances. While drugs such as diazepam and pentobarbital can increase the conductance of some native channels, such properties have not been reported for recombinantly expressed GABA_A receptors. We have recently shown that clustering of GABA_A receptors, induced by the co-expression of the cytoplasmic protein GABARAP in L929 cells, results in conductances significantly greater than 40 pS and that this conductance is increased by both diazepam and pentobarbital (Everitt et al., 2004). In this study we are examining both the physical nature of the clustering of GABA_A receptors and the electrophysiological consequences associated with it.
Organisation and composition of extrasynaptic GABA_A receptors
Our research is directed towards developing an understanding of the relationship between drug action and the organised expression of GABA_A receptors. Using a model system, cultured rat hippocampal neurons, Professor Gage and his group have described the variable conductance of these GABA-activated single channels, from below 10 pS to above 80 pS (Curmi et al., 1993; Birnir et al., 1994; Eghbali, 1997, 2003a,b; Birnir et al., 2000). In a long-standing collaboration with Professor Gage we are exploring the functional consequences of GABA_A receptor clustering and determining whether there are constraints on the subunit composition of GABA_A receptors that participate in clusters. Studies presently are focused on finding out whether the high single-channel conductance displayed by these native extrasynaptic GABA_A receptors, results simply from their being clustered in the membrane. If this were so, it would represent a hitherto undescribed way of regulating GABA_A receptor activity and hence the excitability of neurons.
Molecular mechanisms of ion conduction and gating the GABA_A receptor
GABA_A receptors belong to a superfamily of ligand-gated ion channels and mediate inhibitory neurotransmission in the vertebrate central nervous system by gating Cl- ions through an integral membrane channel. Our interest, at the molecular level, includes identifying specific residues critical for ion conduction through the pore and in the control of gating (opening and closing of the channel).
(a) Molecular components of ion conduction and gating. This work on defining molecular roles for pore-lining residues in gating, ion permeation and protein interactions of the GABA_A receptor combines the use of site-directed mutagenesis of residues in the M2 region with single-channel recordings and analyses the membrane distribution pattern, conductance and kinetic properties of mutant receptors.
(b) Defining the role of the conserved cysteine loop in ligand-gated ion channels.
All ligand-gated ion channel subunits contain a disulphide loop in their extracellular, ligand-binding domain, referred to as the Cys-loop. The precise length and general chemical character of the Cys-loop residues are conserved in these ion channels but not in the soluble ligand-binding protein AChBP, which serves as a model of this domain in the receptor. It is the precise nature of the role of the cysteine loop in ligand-gated ion channels that we are investigating. Specifically, our experiments are directed toward distinguishing between a role of the cysteine loop in the molecular gating mechanism and/ or in the binding of anaesthetics in GABA_A receptors.
(c) Soluble models of ligand-gated ion channels for structural and biochemical analysis The ligand-binding domains of all ligand-gated ion channels have intrinsically the same function, the tightly regulated activation of an ion-selective pore. A fortunate consequence of the modularity of ligand-gated ion channel receptors is that the ligand-binding domain may be expressed in isolation from the rest of the subunit (Tierney & Unwin, 2000). The approaches we employ combine the recombinant expression of soluble ligand-binding domains and X-ray diffraction to determine structure to atomic resolution. The information derived from the X-ray crystallographic structure of the extracellular portion of ligand-gated ion channel receptors will resolve the long-standing issues of neurotransmitter specificity, subunit arrangement around the pentamer and the allosteric processes of activation and desensitisation.

PhD (graduate) students
are trained in advanced techniques of molecular biology, electrophysiology and biophysics. Our group - with the Muscle Physiology Group (Prof Angela Dulhunty)- form the Membrane Biology Program that shares resources and holds meetings and weekly seminars. Scholarships are available on a competitive basis for students to study for a PhD in this program.