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Membrane proteins play a vital role in physiology by controlling the movement of nutrients, waste products and chemical messengers across the cell membrane. Their dysfunction is implicated in a myriad of diseases and they are the target for many therapeutic drugs, but also for a number of drugs of abuse and various toxic compounds. In spite of their importance in biology, membrane proteins are notoriously difficult to crystallize due to their inherent hydrophobicity. As a consequence, there are only a small number of integral membrane protein structures known and there is a large void in our understanding of the molecular mechanisms of membrane protein function. We are particularly interested in neurotransmitter transporters as their modulation has the capacity to selectively manipulate neurotransmitter concentrations and thereby enhance or diminish signalling through particular brain pathways.
We investigate the pharmacology and biochemistry of neurotransmitter transporters in the central nervous system. This involves the use of a combination of recombinant DNA and electrophysiological techniques to investigate the molecular basis for the actions of endogenous modulators as well as novel agents on human neurotransmitter transporters. We are particularly interested in glutamate transporters as dysfunction of the transport processes associated with this key excitatory amino acid has been implicated in the pathogenesis of a number of neurological disorders, including: motor neurone disease, Alzheimer's disease and also ischaemia following a stroke.
We are also interested in the role of glycine transporters in controlling glycine levels in the central nervous system. There are a number of potentially therapeutic drugs, including anti-psychotics and analgesics, which target glycine transporters and we are investigating the molecular basis for the actions of these drugs.
The crystal structures of prokaryotic homologues of both glutamate (GltPh) and glycine (LeuTAa) transporters have recently been determined. These snapshots have provided insights into how the human glutamate and glycine transporters work and also how drugs bind to and affect their function. Our research utilizes the crystal structure of GltPh and LeuTAa to guide the characterization of glutamate and glycine transporters.
To carry out this research we use the following techniques: molecular biology; electrophysiological recordings from Xenopus laevis oocytes expressing recombinant transporters; protein purification and reconstitution into liposomes for functional analysis; X-ray crystallography; computer modeling of protein structures and ligand docking.
Molecular dynamics of glutamate transport
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system and activates a wide range of receptors to mediate a complex array of functions. Extracellular glutamate concentrations are tightly controlled by a family of glutamate transporters expressed in both neurons and glia, which serve to maintain subtly different dynamic signalling systems between neurones. In humans, these transporters are known as Excitatory Amino Acid Transporters (EAATs).
The aim of this project is to develop a structural model for how the EAATs work, and in this way lay the foundations for a more rationale approach to the development of drugs that are both transporter-specific and subtype selective. Such compounds will help to delineate the roles of different transporter subtypes in normal brain functions and also in various neuropathological conditions, such as ischemia following a stroke, Alzheimer’s disease and motor neurone disease.
We use the crystal structure of the prokaryotic aspartate transporter GltPh to direct our research on the human EAATs to try and understand the conformational changes associated with glutamate transport. We are also interested in further understanding the mechanism of GltPh and, in collaboration with Serdar Kuyucak, are using molecular dynamics simulations to probe the substrate/ion binding sites and conformational changes of this archaeal protein.
Figure 1. Permeation pathway of GltPh.
Two of the three protomers of GltPh are shown in the plane of the membrane. Transmembrane domains (TM) 1-6 are in ribbon representation, TM 7, 8, hairpin 1 (HP1) and hairpin 2 (HP2) are shown as cylinders and bound aspartate is shown in stick representation. HP2 (red) serves as an extracellular gate and controls the access of aspartate to its binding site. Sodium ion 2 (purple) serves as a lock on this gate, providing additional energy necessary for its closure.
Below the substrate-binding site is sodium ion 1 (green) and bound solvent (red sphere). The proposed intracellular gate is formed by HP1 (yellow), TM7 (orange) and TM8 (magenta), which are held together by sodium ion 1. The proposed permeation pathway for the substrate is shown as a solid line and motions of HP2 are shown as a dashed double-headed arrow (Boudker, Ryan et al., 2007, Nature, 445, 387-393).
Glycine Transport Inhibitors
Glycine transporters can regulate excitatory and inhibitory neurotransmission in the brain and spinal cord and it has been suggested that inhibitors of glycine transporters may be useful in the treatment of schizophrenia, alcohol addiction and pain.
At present, there are no therapeutic drugs on the market that modulate the activity of glycine transporters. However, a number of glycine transporter 1 (GLYT1) inhibitors are under clinical trials as potential antipsychotics and glycine transporter 2 (GLYT2) inhibitors have been tested in animal models of pain. In this project we will identify novel glycine transport inhibitors and also how these compounds bind to GLYT1 or GLYT2 to better understand the molecular basis for subtype selectivity and specificity of drug actions.
We recently demonstrated that N-arachidonyl-glycine is a potent, selective GLYT2 inhibitor and we are currently screening the activity of related compounds for possible development as a novel class of analgesics. In addition, through collaboration with researchers at the University of Queensland, we are screening spider venoms for inhibitory activity on GLYT1 and GLYT2.
Figure 2. Proposed interactions between N-arachidonyl-glycine and GLYT2.
The structure of LeuTAa with leucine and two sodium ions (purple) bound (PDB: 2A65).
Extracellular loop 2 (EL2, grey), extracellular loop 4 (EL4, blue) and the LeuTAa residues corresponding to the GLYT2 residues I545 (red) and R531 and K532 (orange) shown to interact with N-arachidonyl-glycine are highlighted.
The structure of NAGly is also presented at the approximate scale relative to the structure of LeuTAa (Edington et al., 2009, The Journal of Biological Chemistry, in press).
National Health and Medical Research Council
Australian Research Council
Chuck Bailey, Jeff Holst, Mika Jormakka, John Rasko (Centenary Institute)
Olga Boudker (Cornell University)
Serdar Kuyucak (School of Physics, University of Sydney)