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We study the structure and function of biomolecules using atomic force microscopy (AFM). AFM enables the imaging of individual molecules, such as proteins and DNA, under near-physiological conditions (e.g. under fluid).
The structure of ionotropic receptors
Ionotropic receptors are involved in fast synaptic transmission. These receptors are often composed of at least two types of subunit. Although the subunit stoichiometry is sometimes known, the arrangement of subunits around the receptor rosette is usually undetermined. We produce receptors containing epitope-tagged subunits and incubate them with anti-epitope antibodies.
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AFM image showing a P2X2 receptor (large central particle) decorated by two anti-receptor antibodies (smaller particles). The angle between the two antibodies (120°) indicates that the receptor is composed of three subunits. The image is 30 nm square.
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AFM imaging of the receptor-antibody complexes enables us to deduce the structures of the receptors. For instance, we have shown that the P2X 2 receptor assembles as a trimer. In contrast, the P2X 6 subunit cannot oligomerise by itself; however, P2X 2 and P2X 6 form heterotrimers whose stoichiometry depends on the relative expression levels of the two subunits. In addition, we have shown that the 5-HT 3A/B receptor heteromer assembles as a pentamer of subunit stoichiometry 2A:3B and arrangement B-B-A-B-A. Finally, we have shown that the subunit arrangement within the α 4ß 3δ form of the GABA A receptor is αßαδß, counter-clockwise when viewed from the extracellular face of the membrane. GABA A receptors containing δ-subunits, although a minor component of the total GABA A receptor population, have interesting properties, such as an extrasynaptic location and a high sensitivity to GABA. They are also the target for several general anaesthetics, and have been associated with conditions such as epilepsy and pre-menstrual syndrome. They are therefore attractive targets for drug development. Mike talks about this work here.
Currently, we are studying the subunit arrangement in NMDA receptors. These receptors are widely expressed in the central nervous system, and play a major role in excitatory synaptic transmission; furthermore, because of their involvement in a number of neurological disorders, they also represent significant therapeutic targets. There are various NMDA receptor subtypes, which differ in their subunit composition, and in their biophysical and pharmacological properties. Evidence is emerging that these different subtypes are involved in specific brain disorders.
The structure of ion channels
We are looking at the structure and behaviour of two types of ion channel: the acid-sensing ion channel (ASIC) and polycystin 2.
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AFM image demostrating the trimeric structure of an acid-sensing ion channel (ASIC).
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ASICs are activated by extracellular protons, and are responsible for acid-evoked currents in many neurons of the peripheral and central nervous systems. They appear to play diverse roles in functions such as nociception, learning and memory, and in pathological conditions such as ischemic stroke. We are studying the interactions of ASICs with other membrane proteins, such as the sigma receptor, and visualizing structural changes that they undergo in response to changes in pH.
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Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest inherited human disorders, with a population prevalence of over 1:1,000 in all ethnic groups. There are over 50,000 affected and at-risk individuals in the UK, and up to 12.5 million worldwide. The disorder is a leading cause of end-stage renal failure, and accounts for ~6% of patients on renal replacement programmes in the UK. ADPKD is characterised by the progressive loss of normal renal parenchyma secondary to the development of multiple fluid-filled cysts derived from renal tubular epithelial cells. It is caused by mutations in two genes, PKD1 and PKD2, whose protein products, polycystin-1 and polycystin-2 (or TRPP2) form a mechanosensory Ca2+-permeable ion channel complex. This complex transduces extracellular mechanical stimuli via the renal primary cilium and regulates multiple intracellular Ca2+-sensitive signalling pathways. TRPP2, a noncanonical TRP channel, may also interact with other TRP channel subunits. The goals of our work are to define: (1) the interactions of TRPP2 with two other members of the TRP channel superfamily, TRPC1 and TRPV4; (2) the interaction between polycystin-1 and TRPP2; (3) the interaction of TRPP2 with the syntaxins; and (4) the effects of pathogenic PKD2 mutations on channel assembly and function.
The behaviour of proteins involved in neurotransmitter release and membrane recapture
We are investigating the interactions of a number of proteins with lipid bilayers. These proteins include synaptotagmin, the Ca2+ sensor for neurotransmitter release, endophilin, a protein involved in membrane recapture at the nerve terminal, and botulinum neurotoxin B, which blocks neurotransmitter release by cleaving the SNARE protein, synaptobrevin. We are also looking at the structure of the SNARE complex, which mediates membrane fusion during exocytosis at the nerve terminal. We are interested in the structures that these proteins adopt when associated with the bilayer, and also in the ways in which they perturb the structure of the bilayer.
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AFM image of synaptotagmin (white particles) in association with a lipid bilayer (orange surface).
Between successive images one synaptotagmin
particle dissociates, leaving behind an indentation in the bilayer (arrow).
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Current lab members
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Swetha Suresh, Ian Fyfe, below Andy Stewart using the atomic force microscope
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Peter Oatley and Ligia Antonio
Our work is funded by the Biotechnology and Biological Sciences Research Council, the Wellcome Trust and Kidney Research UK.
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Kobori, T., Smith, G.D., Sandford, R. and Edwardson, J.M. (2009) The transient receptor potential (TRP) channels TRPP2 and TRPC1 form a heterotetramer with a 2:2 stoichiometry and an alternating subunit arrangement. J. Biol. Chem. (in press; doi:10.1074/jbc.M109.060228)
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Neaves, K.J., Huppert, J.L., Henderson, R.M., and Edwardson, J.M. (2009) Direct visualization of G-quadruplexes in DNA using atomic force microscopy. Nucl. Acids Res.37, 6269-6275
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Shahin, V., Datta, D., Hui, E., Henderson, R.M., Chapman, E.R. and Edwardson, J.M. (2008) Synaptotagmin perturbs the structure of phospholipid bilayers. Biochemistry 47, 2143-2152
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Barrera, N.P., Betts, J., You, H., Henderson, R.M., Martin, I.L., Dunn, S.M.J. and Edwardson, J.M. (2008) Atomic force microscopy reveals the stoichiometry and subunit arrangement of the α4β3δ GABAA receptor. Mol. Pharm. 73, 960-967
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Barrera, N.P. and Edwardson, J.M. (2008) The subunit arrangement and assembly of ionotropic receptors. Trends Neurosci. 31, 569-576
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Barrera, N.P., Henderson, R.M., Murrell-Lagnado, R.D. and Edwardson, J.M. (2007) The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels. Biophys. J. 93, 505-512
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Barrera, N.P., Herbert, P., Henderson, R.M., Martin, I.L and Edwardson, J.M. (2005) Atomic force microscopy reveals the stoichiometry and subunit arrangement of 5-HT3 receptors. Proc. Natl. Acad. Sci. U.S.A. 102, 12595-12600
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Barrera, N.P., Ormond, S.J., Henderson, R.M., Murrell-Lagnado, R.D. and Edwardson, J.M. (2005) AFM imaging demonstrates that P2X2 receptors are trimers, but that P2X6 receptor subunits do not oligomerize. J. Biol. Chem. 280, 10759-10765
PubMed link to all Mike Edwardson’s publications
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Selected Publications