Molecular analysis of AMPA-specific receptors: subunit composition, editing, and calcium influx determination in small amounts of tissue

Abstract Glutamate activates three distinct classes of ionotropic receptors: AMPA, kainate and NMDA. AMPA receptors (AMPARs) are of particular importance as they mediate the majority of fast excitatory synaptic transmission and are implicated in a variety of neurological disorders [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123–139]. Functional AMPARs are believed to be a heteromer comprising a combination of four closely related subunits, GluRs1–4 [B. Bettler, C. Mulle, AMPA and kainate receptors, Neuropharmacology 34 (1995) 123–139]. Diversity of AMPARs is obtained through multiple combinations of AMPAR subunits, by alternative splicing of subunits at the flip / flop and/or C-terminal sites, and by mRNA editing of a single amino acid at multiple sites [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA–AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851–853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop : a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580–1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. The subunit combination, editing status, and splice variant expression have profound effects on channel kinetics and can serve as predictors of the channel's properties [M. Hollmann, M. Hartley, S. Heinemann, Ca2+ permeability of KA–AMPA-gated glutamate receptor channel depends on subunit composition, Science 252 (1991) 851–853; B. Sommer, K. Keinanen, T.A. Verdoorn, W. Wisden, N. Burhashev, A. Herb, M. Kohler, T. Takagi, B. Sakmann, P.H. Seeburg, Flip and flop : a cell-specific functional switch in glutamate-operated channels in the CNS, Science 249 (1990) 1580–1585; B. Sommer, M. Kohler, R. Sprengel, P.H. Seeburg, RNA editing in brain controls a determinant of ion flow in glutamate-gated channels, Cell 67 (1991)]. In this manuscript, we detail procedures for profiling AMPAR composition, namely: relative subunit ratios, expression of flip / flop isoforms, Q/R and R/G editing status, and Ca 2+ permeability using small amounts of cDNA from identified cell populations. Themes : Development and regeneration Topics : Neurotransmitter systems and channels