The GABAA receptor alpha 1 subunit Pro174-Asp191 segment is involved in GABA binding and channel gating.
The GABA-binding site undergoes structural rearrangements during the transition from agonist binding to channel opening. To define possible roles of the GABA(A) receptor alpha(1) subunit Pro(174)-Asp(191) segment in these processes, we used the substituted cysteine accessibility method to characterize this region. Each residue was individually mutated to cysteine, expressed with wild-type beta(2) subunits in Xenopus laevis oocytes, and examined using two-electrode voltage clamp. Most mutations did not alter GABA EC(50) values. The D183C mutation produced a 7-fold reduction in GABA sensitivity. There were no significant changes in the K(I) values for the competitive antagonist, SR-95531. N-Biotinylaminoethyl methanethiosulfonate modified P174C-, R176C-, S177C-, V178C-, V180C-, A181C-, D183C-, R186C- and N188C-containing receptors. The pattern of accessibility suggests that this protein segment is aqueous-exposed and adopts a random coil conformation. Both GABA and SR-95531 slowed covalent modification of V178C, V180C, and D183C, indicating that these residues may line the GABA-binding site. Further, pentobarbital-induced channel activation accelerated modification of V180C and A181C and slowed the modification of R186C, suggesting that this region of the alpha(1) subunit may act as a dynamic element during channel-gating transitions.
Newell
J Glen
JG
Department of Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Czajkowski
Cynthia
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010424:Pentobarbital / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D010446:Peptide Fragments; D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D010424:Pentobarbital