GABA(A) receptor beta 2 Tyr97 and Leu99 line the GABA-binding site. Insights into mechanisms of agonist and antagonist actions

J Biol Chem. 2002 Jan 25;277(4):2931-7. doi: 10.1074/jbc.M109334200. Epub 2001 Nov 15.

Abstract

The identification of residues that line neurotransmitter-binding sites and catalyze allosteric transitions that result in channel gating is crucial for understanding ligand-gated ion channel function. In this study, we used the substituted cysteine accessibility method and two-electrode voltage clamp to identify novel gamma-aminobutyric acid (GABA)-binding site residues and to elucidate the secondary structure of the Trp(92)-Asp(101) region of the beta(2) subunit. Each residue was mutated individually to cysteine and expressed with wild-type alpha(1) subunits in Xenopus oocytes. GABA-gated currents (I(GABA)) were measured before and after exposure to the sulfhydryl reagent, N-biotinylaminoethyl methanethiosulfonate (MTS). V93C, D95C, Y97C, and L99C are accessible to derivatization. This pattern of accessibility is consistent with beta(2)Val(93)-Leu(99) adopting a beta-strand conformation. Both GABA and SR95531 protect Y97C and L99C from modification, indicating that these two residues line the GABA-binding site. In D95C-containing receptors, application of MTS in the presence of SR95531 causes a greater effect on I(GABA) than MTS alone, suggesting that binding of a competitive antagonist can cause movements in the binding site. In addition, we present evidence that beta(2)L99C homomers form spontaneously open channels. Thus, mutation of a binding site residue can alter channel gating, which implies that Leu(99) may be important for coupling agonist binding to channel gating.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Carrier Proteins / chemistry
  • Cysteine / chemistry
  • DNA, Complementary / metabolism
  • Dimerization
  • Dose-Response Relationship, Drug
  • GABA Antagonists / pharmacology
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Oocytes / metabolism
  • Patch-Clamp Techniques
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Pyridazines / pharmacology
  • Rats
  • Receptors, GABA-A / chemistry*
  • Receptors, GABA-A / metabolism
  • Xenopus
  • gamma-Aminobutyric Acid / chemistry*
  • gamma-Aminobutyric Acid / metabolism*

Substances

  • AChBP protein, Lymnaea
  • Carrier Proteins
  • DNA, Complementary
  • GABA Antagonists
  • Pyridazines
  • Receptors, GABA-A
  • gamma-Aminobutyric Acid
  • gabazine
  • Cysteine