Studentship Proposal by hdh16952

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									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 α6β342 nAChRs. Unlike other brain nAChRs, α6β342 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, α6β342 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 α6β342 nAChRs. This is mostly due to the
scarcity of naturally expressed α6β342 nAChRs. Furthermore, the well-established strategy of studying
receptors by using recombinant receptors assembled in expression systems has not been successfully
applied to α6β342 nAChRs. This is because α6β342 nAChRs are composed of four different types of
subunits, which when expressed together in cell expression systems produce a multiplicity of receptors
including 42, 62, 623, 423 and α6β342 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 α6β342 nAChR that will be used to
express human α6β342 nAChRs with fixed subunit composition and arrangement in Xenopus oocytes; b)
use the concatenated α6β342 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 α6β342 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 α6β342 nAChR. The strategy to construct concatenated α6β342 nAChRs will be
similar to that we have used to engineer 42 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 62-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 α6β342 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
α6β342 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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