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An investigation into the kinetic properites of NMDA receptors using chimaeric channel constructs

Reference	BB/D001978/1
Principal Investigator / Supervisor	Professor David Wyllie
Co-Investigators / Co-Supervisors	Dr Philip Chen
Institution	University of Edinburgh
Department	Neuroscience
Funding type	Research
Value (£)	128,098
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	30/11/2007
Duration	26 months

Abstract

This proposal continues our structure-function investigations of the NMDA receptor. This receptor-channel mediates the slow component of the glutamatergic excitatory postsynaptic current and as well as subserving many physiological and patho-physiological processes in the CNS. NMDA receptors require that two ligands are bound for its activation. Glycine binds to NR1 subunits while glutamate binds to NR2(A-D) subunits and functional NMDA receptors exist as hetero-oligomers containing two NR1 and two NR2 subunits. It is the NR2 subunits that control many of the different biophysical properties seen amongst NMDA receptor subtypes. Glycine and glutamate binding sites are located in NR1 and NR2 NMDA subunits respectively and residues forming these sites are found preceding the M1 region (S1 domain) and between the M3 and M4 regions (S2 domain). Most of our understanding of ligand interactions with iGluRs has come from studies of the AMPA GluR2 S1S2 crystal structure which show that the S1 and S2 regions form a bilobar structure that closes once agonists bind. The crystal structure of the NR1 S1S2 binding site has recently been described. Although these structures only describe the S1 S2 regions in isolation they have provide us with a glimpse into the mechanism by which glutamate may bind at NR2 subunits. For ligand-gated ion channels their physiological roles are determined, in part, by the duration for which agonist remains bound. For glutamatergic transmission the kinetic behaviour of NMDA receptors determines, in part, the slow depolarization of a neurone, facilitating synaptic integration, co-incidence detection for types of Hebbian plasticity and has a major influence on the temporal characteristics of calcium rises in postsynaptic neurones following activation of excitatory synapses. Thus the duration glutamate remains bound to the NMDA receptor is therefore pivotal to its function as a neurotransmitter. Moreover the factors that control the duration and nature ofNMDA receptor mediated activations are important for developing a kinetic model to describe the physiological behaviour of the channel. Using the information we have gathered studying glutamate binding site point mutations in the NR2A subunit, we plan to extend this by examining the molecular determinants underlying the kinetic behaviour of NMDA receptors. This will involve the examining (i) the pharmacology, deactivation kinetics of macroscopic currents and single-channel properties of chimaeric NMDA receptors where we have swapped functional binding domains between different NMDA receptor subtypes or between AMPA and NMDA receptor subtypes and (ii) the contribution of different NR1 splice variants to the kinetic behaviour of NMDA receptors. This will be achieved through a combination of molecular and electrophysiological techniques including PCR based mutagenesis, two electrode voltage clamp recording, patch-clamp electrophysiology and fast concentration jumps to study the pharmacology, individual single-channel activations and deactivation kinetics of these mutant receptor-channels. Relating structure to function is a major goal for the study of any neurotransmitter receptor. This has best been achieved for ionotropic receptors, perhaps most notably for the nicotinic acetylcholine receptor found at muscle endplate, from the work of Unwin. The recent work of Gouaux and colleagues has provided major insights into aspects of channel gating of glutamate receptors. Our proposal complements these studies by extending our knowledge of NMDA receptor-channels by taking separate functional domains and determining how these structural elements influence kinetic behaviour.

Summary

The human brain contains upwards of 100 billion neurones (nerve cells) which form a network that acts as an 'information superhighway' that continuously sends and receives signals, processing these to control every aspect of our behaviour / from simple, but fundamental, tasks such as breathing and walking to the complex such as perceiving emotion, interpreting our senses and storing and recalling memories. Neurones 'communicate' with each other at specialized sites known as synapses / a site where one neurone releases a chemical (known as a neurotransmitter) that binds to specific proteins (receptors) on the second neurone. There are many different neurotransmitters in the brain but the most common one that causes excitation is glutamate. Glutamate binds to several different types of receptors but one in particular has been the focus for much research over the past 20 years and is known as the NMDA receptor. This receptor plays a very important role in both normal and abnormal brain function. For example, in early life this receptor helps to ensure that the correct wiring pattern is laid down in the developing brain, without it we would be unable to store many types of memories, if it is overactivated it causes epilepsy while following a stroke the glutamate released from dying cells acts on NMDA receptors to cause more neurones to die and its altered function may play a role in such diseases as Alzheimer's, Parkinson's and schizophrenia. The NMDA receptor protein falls into a class of neurotransmitter receptors which contain within their structure a pore which opens when the neurotransmitter binds to it. This pore allows ions to flow into the neurone generating an electrical signal which we can measure and which is essential for the process of synaptic transmission. To complicate things a little more there are four different NMDA receptor subtypes which all are subtley different in their function. We know the amino acid sequence of all NMDA receptor subtypes and because of this we are able to clone the proteins and express them in cells to study their function. Studies from our lab in the past have been aimed at understanding its function at the very basic level and we have done this by making changes to the amino acid sequence of the NMDA receptor (i.e. mutating the protein). Previous work from related proteins has given us a rough idea of how blocks of the receptor, made up of several hundreds of amino acids, allow it to function and we are particularly interested in the parts of the protein that bind glutamate (binding site). It is these parts of the protein that seem to determine how long glutamate remains bound to the receptor (and to a certain extent how long the ion pore will remain open) and this seems to vary amongst the different NMDA receptor subtypes. However as the receptor consists of around 1500 amino acid residues it would be very laborious to individually mutate all of these to try and study the receptor's function. Rather this project aims to swap blocks of one subtype of the NMDA receptor with a different subtype to try and find out what is it about these blocks that make each of the different subtypes have unique properties.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
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	X – not Funded via a specific Funding Scheme

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