Book of abstracts - British Neuroscience Association

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.
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