Distribution and molecular organisation of native kainate receptors in the mammalian central nervous system

Neurotransmitter receptors are proteins that allow cells in the central nervous system to communicate effectively and rapidly. The vast majority of excitatory neurotransmitter receptors in the mammalian brain are activated by the amino acid glutamate. Glutamate is released from neurons in a highly regulated and activity dependent manner. There are several different classes of receptors which respond to glutamate. The least well characterised of these are the kainate receptors (KARs). It is important to gain more information about the precise distribution, molecular and pharmacological properties of KARs, because increased understanding of the mechanisms that regulate them will allow their involvement in normal brain function to be better defined. In addition, KARs have been implicated in neurological disease states and their activation has been shown to be potently neurotoxic. Recently we have developed a range of new reagents (antibodies) and pharmacological tools which created new opportunities for the selective identification, isolation and analysis of KARs. Our research will exploit the availability of these new tools to provide basic information about the distribution and molecular composition of KARs in different functionally important regions of the adult and developing brain. The development of the brain is a complicated process involving millions of connections forming between neurons. The molecular and cellular mechanisms governing the development of neuronal connections are fundamentally important to normal brain function. Our studies will elucidate the role of KARs in neuronal development. Previous studies established that sensory experience (e.g.: visual experience) guides the development of neuronal circuits. We will test the hypothesis that KARs are influenced by visual experience. The results of the proposed studies may help the future development of therapeutic agents such as anticonvulsants for the treatment of diseases like epilepsy and/or neuroprotective drugs that could be of value for treating conditions such as stroke and head trauma.

Kainate receptors (KARs) are ligand gated ion channels that belong to the glutamate receptor family. Recent studies have identified KARs as key players in the modulation of neuronal network activity throughout the CNS. KARs are also involved in neuronal differentiation, synaptic plasticity and CNS disorders. The distribution, developmental profile and molecular organisation of native KARs in neurons are poorly understood. This gap in our knowledge seriously hampers our understanding of the roles of KARs in neuronal signalling mechanisms and functions. The availability of more selective drugs, transgenic mice and electrophysiological studies has started to reveal the pharmacological and functional properties of KARs. However, the understanding of the differential distribution and the region/synapse-specific subunit composition of native KARs remains a major goal in neurosciences. The research proposed here aims to use a combination of recently developed KAR subunit selective immunoreagents, novel pharmacological tools and labelling strategies to gain further basic information about native KARs in the adult and developing brain. The proposed experiments will focus on the identification of the regional, cellular and subcellular distribution of KAR subunit proteins, subunit composition of native KARs and properties of native KARs targeted by the recently developed GluR5 subunit selective antagonists (UBP310 and ACET) in the CNS. We will use a combination of pharmacological, biochemical, autoradiography and immunohistochemical approaches to investigate native KARs in the brain and in primary neuronal cultures. A new radiolabelled GluR5 specific antagonist [3H]UBP310 will be synthesised which will allow ligand-binding and autoradiography studies. Our experimental strategy is suitable for the parallel quantitative analysis of subunit protein expression and the distribution of [3H]UBP310 binding activity in different brain regions. Based on the analysis of the X-ray crystal structure of the GluR5 ligand binding domain and docked antagonist ACET, we will synthesise a biotin-conjugated and azido group containing version of ACET without loss of affinity for GluR5. This ligand will be used for the covalent photo-labelling, localisation and affinity chromatography separation of GluR5 subunit-containing KAR complexes in native neurons for subsequent biochemical analysis. The proposed studies will allow us to gain some understanding of the rules neurons follow during the expression, assembly and targeting of kainate receptors, and the mechanism by which KARs are modified during devlopment and influenced by visual experience in the CNS.