Molecular analysis of AMPA-specific receptors: subunit composition, editing, and calcium influx determination in small amounts of tissue.
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 Ca2+ permeability using small amounts of cDNA from identified cell populations.
Lee
J C
JC
Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.
Greig
A
A
Ravindranathan
A
A
Parks
T N
TN
Rao
M S
MS
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002642:Chick Embryo; D003035:Cobalt / Q000493:pharmacokinetics; D018076:DNA, Complementary / Q000235:genetics; D008969:Molecular Sequence Data; D012326:RNA Splicing / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000032:analysis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
D018076:DNA, Complementary; D018091:Receptors, AMPA; D003035:Cobalt; D002118:Calcium