Purification and In Vitro Functional Analysis of Glutamate Receptor

Abstract

Ionotropic glutamate receptors are predominantly localized to neuronal synapses and are principal mediators of excitatory neurotransmission in the brain. Receptor function is regulated by multiple factors including ligands, ions, pH, posttranslational modification, subunit composition, RNA editing, and elements of the lipid environment. Analysis of channel function in native membranes has provided much quantitative information on these various regulatory features, however, investigation of lipid regulation is seriously hampered by an inability to control membrane composition in living cells. Although an inhibitory effect of free polyunsaturated fatty acids on GluK2(R) activation has been demonstrated, the specific bulk lipid requirements for normal channel operation, as well as susceptibility to fatty acid modulation, are unknown. To address this, we aim to purify rat kainate receptor 2 (GluK2), to reconstitute the purified protein, and to assess channel function under different defined lipid environments. Using Saccharomyces cerevisiae as an expression host, we have been able to express wild type GluK2 with either Gln or Arg at the editing site, and with different tags. In addition, we have expressed modified forms of GluK2 including an amino terminal domain (ATD) deletion mutant, and a double point mutation (Y590C/L572C) that allows disulfide crosslinking between ligand binding domain (LBD) dimers. We have succeeded in purifying wild type GluK2(R) through Flag affinity and subsequent size exclusion chromatography after solubilizing the protein using the detergent Foscholin-14 (F14). A major band at the expected monomer size of ∼100kDa and several bands at lower molecular weights are resolved on 1D SDS PAGE and are confirmed to be GluK2 through mass spectrometry (MS) analysis. MS analysis also reveals that a fraction of the heterologously expressed protein in yeast maintains a signal sequence at the N-terminus and is phosphorylated on a presumed extracellular residue, indicating misoriented topology.