Purification and Reconstitution of the AMPA Receptor

Y.C. Chang, I.F. Peng, T.Y. Wu, Y.M. Zhou and H.M Chang

Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, 30043, R.O.C..

The AMPA receptor was purified from the synaptic junctions of pig cerebral cortex by a combination of anion-exchanging chromatography, wheat-germ-agglutinin affinity chromatography and sucrose density gradient centrifugation. SDS-PAGE analysis revealed that the resultant preparation consisted of a single protein of 106 kDa, which was recognized by antibodies to rat GluR1 and GluR2/3 subunits, but not recognized by the antibodies to rat NR1 or GluR6/7 subunits. Western blot analysis performed under non-reducing conditions revealed that the 106 kDa protein of the isolated preparation was cross-linked into oligomers up to tetramers by disufide bonds. Treatments of the purified AMPA receptor with a cross-linking reagent, N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), lead to significant increases in the dimer and tetramer bands and decreases in the monomer and trimer bands. The AMPA receptor in the purified preparation co-migrated with the AMPA receptors whose protein mass was estimated to be around 350 kDa in sucrose density gradient centrifugation analyses. These observations indicate that the purified AMPA receptors are tetrameric assemblies of subunits of 106 kDa in size. The purified AMPA receptor was then reconstituted into liposomes made from soybean phosphatidylcholine and then into artificial lipid bilayers at the tip of patch-clamp electrodes. In the presence of kainate, single-channel currents across the lipid bilayer with the chord conductance at -150 mV in the range between 7 and 25 pS were recorded. These kainate-activated currents were not activated by NMDA. These observations indicate that the AMPA receptor purified in this study still retains its capacity to gate currents in response to pharmacological activation and block. Because our biochemical analyses reveal that the purified AMPA receptors are tetramers, these observations also suggest that four AMPA receptor subunits are sufficient to form a functional AMAP receptor channel.