Detection and binding properties of GABA(A) receptor assembly intermediates.
Density gradient centrifugation of native and recombinant gamma-aminobutyric acid, type A (GABA(A)) receptors was used to detect assembly intermediates. No such intermediates could be identified in extracts from adult rat brain or from human embryonic kidney (HEK) 293 cells transfected with alpha(1), beta(3), and gamma(2) subunits and cultured at 37 degrees C. However, subunit dimers, trimers, tetramers, and pentamers were found in extracts from the brain of 8-10-day-old rats and from alpha(1)beta(3)gamma(2) transfected HEK cells cultured at 25 degrees C. In both systems, alpha(1), beta(3), and gamma(2) subunits could be identified in subunit dimers, indicating that different subunit dimers are formed during GABA(A) receptor assembly. Co-transfection of HEK cells with various combinations of full-length and C-terminally truncated alpha(1) and beta(3) or alpha(1) and gamma(2) subunits and co-immunoprecipitation with subunit-specific antibodies indicated that even subunits containing no transmembrane domain can assemble with each other. Whereas alpha(1)gamma(2), alpha(1)Ngamma(2), alpha(1)gamma(2)N, and alpha(1)Ngamma(2)N, combinations exhibited specific [(3)H]Ro 15-1788 binding, specific [(3)H]muscimol binding could only be found in alpha(1)beta(3) and alpha(1)beta(3)N, but not in alpha(1)Nbeta(3) or alpha(1)Nbeta(3)N combinations. This seems to indicate that a full-length alpha(1) subunit is necessary for the formation of the muscimol-binding site and for the transduction of agonist binding into channel gating.
Section of Biochemical Psychiatry, University Clinic for Psychiatry, A-1090 Vienna, Austria.
T
T
Klausberger
N
N
Ehya
K
K
Fuchs
T
T
Fuchs
V
V
Ebert
I
I
Sarto
W
W
Sieghart
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D005442:Flumazenil / Q000493:pharmacokinetics; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney; D046911:Macromolecular Substances; D009118:Muscimol / Q000493:pharmacokinetics; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D011869:Radioligand Assay; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D046911:Macromolecular Substances; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D014316:Tritium; D009118:Muscimol; D005442:Flumazenil