Nigel Groome PhD Research Studentship Engineering and functional characterisation of nicotinic receptors containing the 6 subunit Supervisor: Prof. Isabel Bermudez Eligibility: Applicants require a good Honours degree (2.1 or equivalent) and either have been educated to degree level through the medium of English or have TOEFL 600 (250) / IELTS 7 or equivalent. Home, EU and international students are eligible. Start date: September 2009 Value p.a. £13,000 bursary & fees Closing Date: 9th March with interviews late March Project: Nicotinic acetylcholine receptors (nAChRs) are proteins that play critical physiological roles throughout the brain (e.g., cognition, memory, analgesia, mood) and body (e.g., muscle contraction, cardiovascular tone, etc) by mediating cholinergic excitatory neurotransmission, modulating the release of neurotransmitters, and having longer-term effects on, for example, gene expression and cellular connections. nAChRs exist as a family of subtypes in the broader superfamily of ligand-gated ion channels. Each nAChR subtype is composed as a pentamer of subunit building blocks, and is defined by its subunit composition. Mammalian nAChR subunits are derived from a family of sixteen different genes (α1-α7, α9- α10, β1-β4, γ, δ and ε) and have distinctive distributions (Lukas et al., 1999). This proposal is concerned with α6β342 nAChRs. Unlike other brain nAChRs, α6β342 nAChRs have a very restricted distribution in the brain. They are predominantly being expressed in midbrain, dopaminergic neurons thought to constitute important elements in pleasure-reward systems, as well as in motor control. As might be expected from this distribution, α6β342 nAChRs are implicated in drug dependence and movement disorders such as Parkinson’s disease and are thus validated drug discovery targets for these brain diseases. In comparison to other type of nAChRs, there has been little progress in the elucidation of the functional and structural properties of α6β342 nAChRs. This is mostly due to the scarcity of naturally expressed α6β342 nAChRs. Furthermore, the well-established strategy of studying receptors by using recombinant receptors assembled in expression systems has not been successfully applied to α6β342 nAChRs. This is because α6β342 nAChRs are composed of four different types of subunits, which when expressed together in cell expression systems produce a multiplicity of receptors including 42, 62, 623, 423 and α6β342 nAChRs. An alternative approach that circumvents the problems of expression of multiple receptor types is to bridge the subunits by synthetic linkers into pentameric concatamers. There truly is no other way to specify subunit composition and subunit arrangement for heterologously expressed nAChRs composed of more than one kind of subunit. This strategy has been used by my group to produce functional expression of (4)2(2)3 nAChR and (4)3(2)2 nAChRs whose properties are similar to those of their native counterparts (Carbone et al., 2009). Aims of the proposal The specific aim of this proposal is a) to construct a model of the α6β342 nAChR that will be used to express human α6β342 nAChRs with fixed subunit composition and arrangement in Xenopus oocytes; b) use the concatenated α6β342 nAChR to characterize the functional properties of the receptor and identify the structural determinants of those properties. The project will be carried out in collaboration with Professor Ron Lukas and Dr Paul Whiteaker from the Barrow Institute of Neurology, Phoenix, Arizona, who are internationally known for their expertise on α6β342 nAChRs and expression of nAChRs in cell lines (link to their web site: http://www.thebarrow.org/Research_and_Clinical_Trials/Basic_Research_Laboratories/Neurochemistry_La boratory/index.htm). Methods Construction of α6β342 nAChR. The strategy to construct concatenated α6β342 nAChRs will be similar to that we have used to engineer 42 nAChR (Carbone et al., 2009). Briefly, the order of the subunits will be 2-4-2-6-3 and the subunits will be linked by the synthetic linker AGS. These procedures will be carried out using standard molecular biological techniques such as PCR and T4- ligations. Expression of 62-concatenated receptors in Xenopus oocytes. Stage VI oocytes will be injected with cRNA doding for the concatenated receptors as described previously (Moroni et al. 2008; Carbone et al., 2009). Two to seven days after injection, oocytes will be tested for expression of concatenated receptors using standard two-electrode voltage clamping approaches (Moroni et al., 2008). The aim of these experiments will be to establish the basic pharmacological properties of these receptors such as their sensitivity to classical α6β342 nAChR’s agonists (e.g., nicotine). The findings of these studies will be used next to identify the structural determinants of the pharmacological properties of the receptor. These studies will use standard site-directed mutagenesis in combination with electrophysiology. Original contribution to knowledge α6β342 nAChRs are involved in the reward and motor functions of the brain and are centrally involved in drug addiction and Parkinson’s disease. Because of its complex subunit composition, this receptor has been difficult to characterize and as a result we know little about its functional and pharmacological properties. The availability of a receptor with a fixed subunit composition or arrangement will be a valuable contribution to the field. Furthermore, the availability of unambiguous data on the functional and pharmacological properties of this receptor will most certainly contribute to progress in the field of nicotinic receptor basic science and drug discovery. References -Baur R, Minier F, Sigel E. (2006) Febs Letters. 2006;580(6):1616-20. -Carbone AL, Moroni M, Bermudez I (2009) BJP. In Press. -Groot-Kormelink PJ, Broadbent S, Beato M, Sivilotti LG. Molecular Pharmacology. 2006;69(2):558-63. To apply for this studentship, please complete the University's Postgraduate Application Form. Applications should be posted to: Ms Lucy Boyles, Postgraduate Administrator School of Life Sciences Oxford Brookes University Headington Oxford OX3 0BP UK Please email Lucy Boyles or telephone +44 (0)1865 483295 with any administrative queries. N.B. Please note that applications will not be accepted by email.
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