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Photoaffinity Labelled Probes for the GABA-A Receptor

Reference	BB/I016635/1
Principal Investigator / Supervisor	Professor James Baker
Co-Investigators / Co-Supervisors	Dr Lyn jones, Professor Trevor Graeme Smart
Institution	University College London
Department	Chemistry
Funding type	Skills
Value (£)	99,932
Status	Completed
Type	Training Grants
Start date	01/10/2011
End date	30/09/2015
Duration	48 months

Abstract

unavailable

Summary

To understand how receptors are targeted to specific inhibitory synapses in the brain, we need to study the 'life cycle' of native receptors on the surface membrane. GABA-A receptors are inserted and undergo constitutive endocytosis and some recycling back to the surface(1,2), which affects the efficacy of synaptic inhibition(1,3). By using a reporter mutation in the GABA ion channel, we demonstrated for the first time that synaptic GABA-A receptors are rapidly mobile on the surface membrane(4) allowing an exchange with extrasynaptic receptors(5). Although the mobility of synaptic/extrasynaptic receptors can be investigated with increased resolution, these methods require the expression of mutant receptor subunits in neurones. To track the movement of native receptors requires the development of highly-selective ligands that can covalently bind to the receptor and affect its function, so that the movement of receptors can be followed using electrophysiological methods. To investigate native GABAA receptor mobility at synapses, we will design and synthesise photoaffinity labelled antagonists that upon irradiation will bind irreversibly to the receptor causing inhibition. We have chosen gabazine as our template molecule since this inhibitor is specific for GABA-A receptors, has high affinity, and as it has been shown that structural variation on the biaryl core is well tolerated(6). We will synthesise photoaffinity labelled gabazine analogues, varying the nature and position of the photoaffinity label. To achieve this we will use a highly efficient 4-step synthesis employing palladium cross-coupling as the key step. By flash irradiation at precise surface membrane locations in the presence of our gabazine analogues, we will inactivate discrete populations of GABA-A receptors (extrasynaptic and/or synaptic). By monitoring both phasic and tonic GABA currents, we will be able to track the movement of native receptors in synaptic and extrasynaptic membrane domains. Adopting this approach means we neither have to mutate the receptor to incorporate a tag nor express this in neurones; secondly, we can measure the speed of movement of receptors in defined membrane areas by monitoring the recovery of GABA currents following photoactivation. We can then distinguish between lateral mobility and exocytosis of GABA-A receptors using selective inhibitors (eg., botulinum toxins) as well as being able to estimate the mean number of functional receptors at inhibitory synapses and their relative change over time(4). Activity-based proteomic profiling has a variety of powerful applications in drug discovery and chemical biology, including sophisticated measurements of selectivity and target occupancy and biomarker development. Affinity-based proteomics extends the utility of this area, and is of significant interest within both academia and the pharmaceutical industry. Notably there remains a paucity of methods and suitable ligands for probing the proteome of ion channels, a recognised drug target. In this collaboration between UCL and Pfizer we want to develop new affinity-based probes for ion channels, to enable measurements of the functional proteome and to determine target occupancy. The GABA ion channel presents an ideal model system to develop the utility of activity-based chemical probes for proteomic profiling. 1. B. Luscher et. al., Pharmacol. Ther. 102, 195 (2004). 2. T. G. Smart et. al., Nat. Rev. Neurosci. 2, 240 (2001). 3. F. K. Bedford et. al., Nat. Neurosci. 4, 908 (2001). 4. T. G. Smart et. al., Nat. Neurosci. 8, 889 (2005). 5. T. C. Jacob et. al., J. Neurosci. 25, 10469 (2005). 6. C. G. Wermuth et. al., J Med. Chem. 30, 239 (1987).

Committee	Not funded via Committee
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	Training Grant - Industrial Case

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