PMID- 10187791
OWN - NLM
STAT- MEDLINE
DCOM- 19990503
LR  - 20210209
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 15
DP  - 1999 Apr 9
TI  - Stoichiometry of a ligand-gated ion channel determined by fluorescence energy 
      transfer.
PG  - 10100-4
AB  - We have developed a method to determine the stoichiometry of subunits within an 
      oligomeric cell surface receptor using fluorescently tagged antibodies to the 
      individual subunits and measuring energy transfer between them. Anti-c-Myc 
      monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium 
      cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or 
      gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor 
      in intact cells. The maximal fluorescent signal derived from the 
      alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice 
      that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 
      2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was 
      extended using fluorescence energy transfer. Receptors were half-maximally 
      saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were 
      labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy 
      acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 
      occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no 
      significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, 
      indicating the absence of a second gamma-subunit in a receptor monomer. We 
      confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two 
      copies each of the alpha- and beta-subunits and one copy of the gamma-subunit 
      (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have 
      general applicability to other multisubunit cell surface proteins.
FAU - Farrar, S J
AU  - Farrar SJ
AD  - Department of Biochemistry and Molecular Biology, Merck Sharp and Dohme Research 
      Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, United 
      Kingdom.
FAU - Whiting, P J
AU  - Whiting PJ
FAU - Bonnert, T P
AU  - Bonnert TP
FAU - McKernan, R M
AU  - McKernan RM
LA  - eng
PT  - Journal Article
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Ion Channels)
RN  - 0 (Ligands)
RN  - 0 (Organometallic Compounds)
RN  - 0 (Proto-Oncogene Proteins c-myc)
RN  - 0 (Receptors, GABA-A)
RN  - 125433-96-7 (europium(III) trisbipyridine cryptate)
RN  - 40P7XK9392 (Flumazenil)
RN  - 444W947O8O (Europium)
SB  - IM
MH  - Antibodies, Monoclonal/metabolism
MH  - Binding Sites
MH  - Cell Line
MH  - Energy Transfer
MH  - Europium
MH  - Flumazenil/metabolism
MH  - Fluorescent Dyes
MH  - Humans
MH  - *Ion Channel Gating
MH  - Ion Channels/*physiology
MH  - Ligands
MH  - Organometallic Compounds
MH  - Proto-Oncogene Proteins c-myc/immunology
MH  - Receptors, GABA-A/metabolism
MH  - Spectrometry, Fluorescence
EDAT- 1999/04/03 00:00
MHDA- 1999/04/03 00:01
CRDT- 1999/04/03 00:00
PHST- 1999/04/03 00:00 [pubmed]
PHST- 1999/04/03 00:01 [medline]
PHST- 1999/04/03 00:00 [entrez]
AID - S0021-9258(19)73689-8 [pii]
AID - 10.1074/jbc.274.15.10100 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Apr 9;274(15):10100-4. doi: 10.1074/jbc.274.15.10100.