The Preferential Reconstitution Of Ampa Receptor Proteins Into Model Lipid Domains With Cholesterol Studied By Atomic Force Microscopy - an Imaging And Force Spectroscopy Study

Abstract

In our research we have conducted an atomic force microscopy (AFM) study of trafficking-like behaviour of neural receptor proteins into lipid raft-like domains. In our initial research we formed artificial rafts by varying a mixture of four phospholipids found in the synapse in order to mimic a synaptic membrane. The most commonly occurring receptor protein in the central nervous system, the AMPA receptor (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), was then reconstituted into these mixtures. The results show a preferential reconstitution of these membrane proteins into lipid rafts of a certain height. AMPA receptors are implicated in long term potentiation, a process thought to underlie learning and memory, with up-regulation of AMPAR numbers in the post-synaptic membrane possibly being a key component of this process.In order to come closer to the mixtures naturally occurring in the synapse we furthered these studies to incorporate cholesterol. The results were a preferential reconstitution of AMPAR proteins but this time into the low domain when cholesterol is present. These surprising results were better understood when we treated this system as a ternary mixture with gel phase lipids, liquid phase lipids and cholesterol acting as an impurity. We studied the phases in terms of the domain heights as well as their mechanical properties. When cholesterol was present, the protein-deficient high domains were stiffer and more viscous.The lateral extent of the lipid domains is typically ∼100nm, so they have structural similarities with the lipid rafts observed to occur in synaptic membranes, albeit with much simpler composition. Dynamic AFM measurements reveal information about the mobility of receptors within and between domains which may shed light on this process.