Structural and functional characterization of reconstituted alpha − amino − 3 − hydroxy − 5 − methyl − 4 − isoxazole propionic acid receptors

This thesis describes a novel reconstitution of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) for the purposes of structural characterization by atomic force microscopy (AFM) and functional characterization by electrical recordings of lipid bilayers. AMPARs are glutamate gated ion channels, ubiquitous in the vertebrate central nervous system where they mediate fast excitatory neurotransmission. In a healthy brain, AMPARs are involved in memory formation and learning and their dysfunction has been related to numerous neurological disorders such as Alzheimer's disease, epilepsy, schizophrenia and many others. AMPARs were reconstituted at high and low densities. Densely reconstituted samples contained >100 receptors per μm2, a value comparable to the AMPAR density at synapses. This allowed, for the first time, the imaging of full length tetrameric AMPARs in native-like conditions and with clearly assigned domains: the extracellular domains extended 14 nm above the membrane in agreement with electron microscopy (EM) and X-ray crystallography data. Lipid-protein interactions were studied in samples with low protein density with the receptors showing preference for lipids in the liquid crystalline phase. The activity of the reconstituted receptors was confirmed through single-channel recordings. This is the first case in which an AMPAR has been reconstituted and given (a) single-channel recordings with (b) physiologically plausible conductance levels and (c) pharmacological and no-protein controls and (d) structure. As a result, previously reported biochemistry and EM are now for the first time available in concert with AFM and single-channel recordings for a purified AMPAR of known composition.