Abstract
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.
Highlights
The ␥-aminobutyric acid, type A receptor (GABAA receptor)1 is the postsynaptic target for GABA, the major inhibitory neurotransmitter of the mammalian central nervous system
Phe-64 and His-101 on the ␣1 subunit of the GABAA receptor, were identified by photoaffinity labeling studies using [3H]muscimol and [3H]flunitrazepam to contribute to the GABA and benzodiazepine binding sites, respectively [15, 17, 18]. These residues apparently contribute to separate binding pockets for the receptor agonist and allosteric modulator respectively, they are separated by only 37 amino acid residues, and as indicated in Fig. 1, four of these residues are identical among all GABAA receptor subunits, and among all ligand-gated ion channel subunits
The GABAA receptor gene family forms a subset of the ligand-gated ion channel superfamily
Summary
The ␥-aminobutyric acid, type A receptor (GABAA receptor)1 is the postsynaptic target for GABA, the major inhibitory neurotransmitter of the mammalian central nervous system. Phe-64 and His-101 on the ␣1 subunit of the GABAA receptor, were identified by photoaffinity labeling studies using [3H]muscimol and [3H]flunitrazepam to contribute to the GABA and benzodiazepine binding sites, respectively [15, 17, 18]. Co-expression of ␣1, 2, and ␥2 subunits formed functional receptors, which showed [3H]flunitrazepam binding and GABA-activated currents that were enhanced by benzodiazepines2 [21, 22].
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