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29.06 Functional and radioligand binding properties of alternate stoichometries of human α4β2 nAChRs Carbone A L, Moretti M, Moroni M, Zwart R, Gotti C, Bermudez I 1,3,6 School of Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford OX3 OBD, UK, 2,5 CNR, University of Milan, 20129, Milan, 4 Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK α4β2 neuronal nicotinic acetylcholine receptors (nAChRs) are ligandgated ion channels belonging to the Cys-loop superfamily of ligandgated ion channels. Several lines of evidence have shown that heterologously expressed α4β2 nAChRs exist in at least two different stoichiometries, (α4)3(β2)2 and (α4)2(β2)3, that differ considerably in their functional and pharmacological properties. By using radioligand binding assays, site-direct mutagenesis and the voltage-clamp technique we have investigated the contribution of the ligand binding sites of the α4β2 nAChR to the functional differences between the two receptor stoichiometries expressed heterologously in Xenopus oocytes. The two stoichiometries can be expressed in Xenopus oocytes by manipulating the ratio of α4 and β2 subunit cDNAs injected. The functional potency and efficacy of a range of nicotinic ligands at both receptor forms were significantly different. In contrast, radioligand binding studies showed no differences in the ligand binding profile of the α4β2 stoichiometries. Single-point mutants of the amino acids that form the hydrophobic ring of the binding site of the α4β2 nAChR showed that the mutations affect the function of the two receptors in a different manner. In contrast, radioligand binding studies showed that the mutations affect the ligand binding properties of both receptors in the same manner and to the same extent. These results suggest that the differences between the two alternate stoichiometries of the α4β2 nACh receptor may lie at the gating level. Thus, nicotinic agonists and competitive antagonists may bind the two stoichiometries with the same affinity but activate the channels in a different manner. 29.07 Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols Granfeldt D, Sinclair J, Millingen M, Lincoln P, Orwar O Department of Chemical and Biological Engineering, Physical Chemistry, Chalmers University of Technology, SE-412 96 Göteborg, Sweden, , Cellectricon AB, Fabriksgatan 7, SE-412 50 Göteborg, Sweden Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (β-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and β-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptor populations. 29.08 Impaired hippocampal network function in mice expressing a hypomorphic NMDA receptor variant: ultrastructural correlates Peddie C J 1, Schoepfer R 2, Davies H A 1, Kraev I 3, Tigaret C 2, Popov V 1,3, Stewart M G 1 1The Open University, Department of Biological Sciences, Walton Hall, Milton Keynes MK7 6AA, 2University College London, Laboratory for Molecular Pharmacology, Gower Street, London NW7 1AA, 3Institute of Cell Biophysics, Pushchino, Russia, NMDA receptors (NMDARs) mediate a form of coincidence detection crucial to the refinement of activity dependent synaptic connectivity, an important mechanism in putative learning and memory processes, such as long-term potentiation (LTP). Expression of NR1 subunit mRNA containing the N598R mutation (NR1R) generates NMDARs with altered Mg2+ sensitivity and Ca2+ permeability, thereby impaired in coincidence detection ability. Mice expressing the NR1Rneo allele, a hypomorphic variant of the NR1R allele, generate both wild-type NR1 and NR1R mRNAs, in a 95:5 ratio. Consequently, NR1Rneo/+ NMDARs are approximately 90% wild-type,
29.10 Identification of molecular determinants that mediate zinc effects on alternate stoichiometries of the human α4β2 Nicotinic Acetylcholine Receptors Moroni M, Vijayan+ R, Biggin+ P, Carbone* A L, Bermudez I* *Oxford Brookes University, School of Life Sciences.+Department of Biochemistry, University of Oxford Chelatable zinc is present in synaptic vesicles and is co-released with a variety of neurotransmitters during synaptic signalling to most likely modulate the activity of pre- and post-synaptic ligand-gated ion channels. Although the effects of Zn2+ on NMDA, GABA-A, Glycine, P2X and α7 nicotinic acetylcholine (nACh) receptors has been characterised in detail, the effects of Zn2+ on α4β2 nACh receptors have not been examined. In heterologous system, such as Xenopus oocytes, the α4β2 nAChR assembles in two alternate stoichiometries, (α4)2(β2)3 and (α)3(β2)2, that differ in functional sensitivity to the neurotransmitter ACh and sensitivity to up-regulation of receptors number by chronic exposure to nicotine. Here we report the molecular determinants that mediate the Zinc effects on the two α4β2 arrangements. The responses elicited by agonists at the (α)3(β2)2 stoichiometry are potentiated by micromolar zinc concentrations and are inhibited by millimolar zinc concentrations, whereas the (α4)2(β2)3 stoichiometry is only inhibited by zinc. The effects of Zn2+ at both receptors were sensitive to external pH and were abolished by DEPC, suggesting that histidines played a crucial role in chelating Zn2+. Modelling and disruption of putative Zn2+ binding sites by alanine substitution suggest the presence of a potentiating site at the αα interface at the (α)3(β2)2 stoichiometry. Two inhibitory sites lie presumably at the interface between the α and the β subunits at both receptors. The (α4)2(β2)3 stoichiometry is also subject to a voltage dependent inhibition that can be eliminated by mutation of histidine and glutamates on the beta subunits. 29.11 Development of the first anti-mRIC-3 specific antibody: identification of RIC-3 protein in the mammalian brain Rensburg Rv (1), Ennaceur A (2), Chazot P L (1) 1. Centre for Integrative Neuroscience, Durham University; 2. Sunderland Pharmacy School, UK The α7-nACh receptor clearly plays a significant role in human emotional and cognitive behaviours, and is currently a therapeutic target for a range of acute and chronic neuropathologies. Therefore, regulation of functional expression of the α7-nAChR has significant implications. Ric-3 has been recently identified as an obligatory trafficking protein for the α7-nAChR in heterologous expression studies, yet to be confirmed in vivo. In order to address this issue, we have developed a new anti-mRic-3 polyclonal antibody, which we have validated and used to define the anatomy of ric-3 expression in the mammalian brain. Our new anti-mRic-3 antibody has identified a major Mr ~55,000 species based on immunoblotting, using murine brain membranes, which is absent in liver and kidney membranes. This immunoreactive species migrates close to the recombinant mRic-3 expressed in HEK 293 cells and is consistent with a potential dimeric species. Two protein species, Mr ~26,000 and ~50,000 were identified when mRic-3 was expressed alone in HEK293 cells, but when co-expressed in a bicystronic IRES-mediated vector together with α7 nAChR, a specific single Mr ~26,000 species was detected. This may indicate that the α7 nAChR disrupts the putative dimeric Ric-3 structure as part of the interaction and/or trafficking process. This requires further study. Our initial immunohistochemistry findings on mouse brain slices will be reported using this novel immunological probe, and compared in parallel, to FITC α-bungarotoxin binding as a measure of α7- AChR complexes. Funded by the BBSRC (UK), Durham School of Health, ORS and NRF (South Africa). 29.12 Clustering of inhibitory GABA-A receptors by a gephyrin/collybistin complex Saiepour L, Harvey R J, Harvey K Department of Pharmacology, The School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX Gephyrin, a peripheral tubulin-linker protein, is now well established as a key molecule in GABA-A receptor clustering and localization at synapses. Curiously, however, no-one has been able to demonstrate either a direct or indirect interaction between any single GABA-AR subunit (α1-6, β1-3, γ1-3, δ, ε, θ and π) and gephyrin in recombinant systems or in vivo. We have recently reported that a mutation in the src homology 3 (SH3) domain of the RhoGEF collybistin results in the mislocalisation of gephyrin and GABA-ARs in a model of human epilepsy (Harvey et al 2004, J. Neurosci. 24:5816-5826). This led us to consider whether the correct membrane apposition of gephyrin via collybistin activity could be crucial for gephyrin-GABA-AR interactions. To examine this possibility, we fused the GABA-AR α2, β2, β3, γ2S and γ2L subunit intracellular loops to various fluorescent proteins. Surprisingly, when expressed alone in HEK293 cells, HcRed-γ2S and HcRed-γ2L targeted to the submembrane compartment. This property was not shown by HcRed alone or EYFP-α2, ECFP-β2, ECFP-β3, HcRed-β2 or HcRed-β3. This targeting does not appear to be dependent on the γ2S/L splicing, nor palmitoylation. When coexpressed with EGFP-gephyrin alone, none of the GABA-AR fusion proteins showed targeting to large intracellular gephyrin aggregates. However, co-expression of collybistin, EGFP-gephyrin and HcRed-γ2L resulted in a re-distribution of HcRed-γ2L to submembrane areas enriched in EGFP-gephyrin. Our results suggest that correct membrane apposition of the γ2L subunit, gephyrin and collybistin are likely to be vital for GABA-AR-gephyrin interactions. 29.13 Identification of a Maf1 / Macoco myosin-like protein complex important for regulating the number of surface GABAA receptors. Smith K, Lumb M J, Arancibia-Carcamo L, Oliver P, Brandon N J, Moss S J, Kittler J T Department of Physiology and Department of Pharmacology University College London, London, WC1E 6BT, UK, Department of Neuroscience, University of Pennsylvania School of Medicine, MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK Appropriate membrane transport and clustering of gamma amino-butyric acid type A receptors (GABAARs) to cell surface and synaptic sites is critical for neuronal inhibition. These processes are in part controlled by interaction of receptor intracellular domains with proteins in the cytosol. Here we have cloned and characterised a novel myosin like protein complex that associates with GABAAR β-subunit intracellular domains and which consists of two previously uncharacterised mammalian proteins, which we have called Maf1 (Membrane protein associated factor 1) and Macoco (Maf1 associated coiled coil protein). Maf1, a 29kDa protein, interacts directly with GABAAR β-subunits and can, in addition, interact with Macoco, a Maf1 binding protein with a large open reading frame of 913 amino acids. Macoco is rich in coiled coil domains and in addition has a myosin tail and intermediate filament domains suggesting a possible role for Macoco in cytoskeletal transport processes. Using in situ hybridisation and antibody labelling approaches we find both Maf1 and Macoco to be expressed in brain and also show Macoco to be enriched at inhibitory postsynaptic sites. In cultured cortical neurones disruption of the interaction between Maf1 and Macoco by overexpression of the Macoco interacting domain of Maf1 results in a decrease in GABAAR cell surface number, as revealed by surface biotinylation. This suggests that a Maf1/Macoco protein complex may regulate GABAAR localisation and surface stability by linking these receptors to vesicular transport or clustering machinery. Page 47/101 - 10/05/2013 - 11:11:03
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Page 5 and 6: 4.01 The effect of hippocampal slic
Page 7 and 8: 4.09 ß-adrenergic modulation of in
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Page 31 and 32: 18.03 Finding cells for replacement
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Page 41 and 42: 28.05 The effects of the FAAH inhib
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Page 73 and 74: 49.04 Regulation of synaptic functi
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65.08 The anti-inflammatory role of
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68.01 A comparison of carbachol and
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