Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.
Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.
Srinivasan
S
S
Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California 90095-1761, USA.
Nichols
C J
CJ
Lawless
G M
GM
Olsen
R W
RW
Tobin
A J
AJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D018755:GABA Agonists / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D008969:Molecular Sequence Data; D009118:Muscimol / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry*
D018755:GABA Agonists; D011963:Receptors, GABA-A; D009118:Muscimol; D010649:Phenylalanine; D014364:Tryptophan; D005998:Glycine