Neuronal and glial AMPARs: auxiliary subunits as targets for ion channel regulation

The functions of our brain rely on the chemical communication that takes place between its myriad cells. One of the most important of these chemicals is glutamate, a neurotransmitter responsible for a majority of fast excitatory signaling. Glutamate acts by binding to receptor proteins at points of contact between nerve cells, and most key features of this excitatory signaling are shaped by the basic properties of one specific class of glutamate receptor - the AMPA receptors (AMPARs). AMPARs are ubiquitous; their activity is integral to all fundamental brain functions, including the processes of sensation, thought, emotion and memory. Because of this, dysfunction of AMPARs causes serious neurological disorders. In particular, one subclass of AMPARs that allow the entry of calcium ions into cells is implicated in the aetiology of several conditions, including multiple sclerosis, motor neuron disease, epilepsy, brain damage following stroke or during birth, and brain tumor growth. As AMPARs play a key role in normal cognition, they are also targets for drug treatments aimed at enhancing cognitive function, notably in neurodegenerative conditions such as Parkinson's and Alzheimer's disease.

Understanding AMPARs is essential if we are to dissect their role in disease pathogenesis. Specifically, there is an urgent need to determine the principles that govern AMPAR function at a cellular and molecular level. Recently, our understanding of these processes advanced significantly, with the identification of proteins that directly associate with AMPARs and dictate their properties and behaviour. Two families of proteins have received particular attention - the transmembrane AMPAR regulatory proteins (TARPs) and the cornichons (CNIHs). Our research is aimed at understanding how these 'auxiliary subunits' control the function of AMPA receptors, with the goal of identifying potential avenues for therapeutic intervention. We will use a combination of proven electrophysiology, imaging and molecular approaches to address three specific goals: (1) to reveal how different auxiliary proteins affect the way in which endogenous molecules and exogenous drugs block or potentiate different classes of AMPARs; (2) to identify the specific auxiliary molecules important for the regulation of calcium-permeable AMPARs, and (3) to elucidate the role played by auxiliary proteins in AMPAR changes thought to underlie pathological conditions involving nerve cells and glia (inflammatory pain and white matter damage).

Potential applications and benefits of this research stem from the fact that it will provide fundamental information about the regulation of an important class of glutamate receptors in nerve cells and glia. It will consolidate our understanding of normal brain function and highlight strategies for treatment of debilitating conditions. By establishing how different auxiliary proteins affect the pharmacology of specific AMPAR subtypes it has the potential to inform new efforts in drug discovery.

We will examine how pharmacological, biophysical and molecular properties of neuronal and glial AMPARs are regulated by TARP and cornichon (CNIH) proteins. The identity and role of these auxiliary subunits will be dissected by comparing recombinant and native AMPARs, by examining mutant and transgenic mice, and by targeted molecular intervention to manipulate protein expression or structure. We will use: (1) recombinant AMPARs expressed with specific TARPs and/or CNIHs in tsA201 cells; (2) identified neurons and glia in dissociated cultures, prepared from wild-type (WT) and mutant or transgenic mice; (3) neurons and glia in acute brain slices.

We will use patch-clamp methods to record pharmacologically isolated AMPAR-mediated macroscopic and single-channel currents, and synaptic currents. We will determine defining kinetic properties, voltage-dependence, calcium-permeability and conductance of recombinant, synaptic and extrasynaptic receptors, as well as the efficacy of blockers and allosteric potentiators. We will use a variety of molecular approaches, and antibody-linked quantum dot single particle tracking to explore the influence of specific auxiliary subunits on AMPAR targeting/trafficking. In collaboration with the groups of Dickenson (UCL) and Diamond (NIH) we will examine the contribution of auxiliary subunits to the properties and regulation of calcium-permeable AMPARs in retinal amacrine cells, and in the processing of nociceptive signals in dorsal horn of the spinal cord. Roger Nicoll (UCSF) will collaborate in provision of TARP knockout mice, and related experiments. Experiments using lentiviral constructs will be carried out in collaboration with Stephanie Schorge (UCL). Our research will provide fundamental information about the regulation of AMPARs in neurons and glia. It will contribute to our understanding of normal brain function, highlight potential strategies for treatment of debilitating conditions and inform new efforts in drug discovery.

Academic impact: this work has the potential to provide new knowledge and scientific advancement. The beneficiaries of this research will be medical researchers (including those in university departments worldwide) and members of the pharmaceutical industry. By providing a clearer understanding of the impact of auxiliary proteins on the mechanisms of block of calcium-permeable AMPARs and identifying auxiliary AMPAR subunits involved in the regulation and targeting of CP-AMPARs, our work may held identify specific research avenues that will lead to strategies to limit calcium entry and cell damage in diseases where this is a central issue. Similarly, understanding the influence of auxiliary proteins on the efficacy and selectivity of AMPAR potentiators may aid the identification of strategies or drugs to enhance cognition in disease states. In addition, academic benefit may come from our development of any new methodologies and techniques to achieve our goals. In the broader sense, our work will also be of benefit to neuroscience researchers wishing to understand normal brain function. Immediate benefits (through exchange of scientific knowledge) are likely to accrue in the short to medium term, and certainly within the lifetime of the project. The programme of research will also have capacity-building impact through the training of highly skilled researchers, who will acquire specific competencies and broader transferable skills.

Economic and societal impact: Given an ageing population, neurological conditions will assume an ever-growing economic and social burden. In the long term, by informing drug development or otherwise helping to facilitate rational approaches to therapy, our studies may ultimately lead to enhanced health and well-being.