Agonist- and antagonist-induced conformational changes of loop F and their contributions to the rho1 GABA receptor function

J Physiol. 2009 Jan 15;587(1):139-53. doi: 10.1113/jphysiol.2008.160093. Epub 2008 Nov 17.

Abstract

Binding of gamma-aminobutyric acid (GABA) to its receptor initiates a conformational change to open the channel, but the mechanism of the channel activation is not well understood. To this end, we scanned loop F (K210-F227) in the N-terminal domain of the rho1 GABA receptor expressed in Xenopus oocytes using a site-specific fluorescence technique. We detected GABA-induced fluorescence changes at six positions (K210, K211, L216, K217, T218 and I222). At these positions the fluorescence changes were dose dependent and highly correlated to the current dose-response, but with lower Hill coefficients. The competitive antagonist 3-aminopropyl(methyl)phosphinic acid (3-APMPA) induced fluorescence changes in the same direction at the four middle or lower positions. The non-competitive antagonist picrotoxin blocked nearly 50% of GABA-induced fluorescence changes at T218 and I222, but only <20% at K210 and K217 and 0% at K211 and L216 positions. Interestingly, the picrotoxin-blocked fraction of the GABA-induced fluorescence changes was highly correlated to the Hill coefficient of the GABA-induced dose-dependent fluorescence change. The PTX-insensitive mutant L216C exhibited the lowest Hill coefficient, similar to that in binding. Thus, the PTX-sensitive fraction reflects the conformational change related to channel gating, whereas the PTX-insensitive fraction represents a binding effect. The binding effect is further supported by the picrotoxin resistance of a competitive antagonist-induced fluorescence change. A cysteine accessibility test further confirmed that L216C and K217C partially line the binding pocket, and I222C became more exposed by GABA. Our results are consistent with a mechanism that an outward movement of the lower part of loop F is coupled to the channel activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cysteine / chemistry
  • Female
  • Fluorescent Dyes
  • GABA-B Receptor Agonists
  • GABA-B Receptor Antagonists
  • Humans
  • In Vitro Techniques
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oocytes / drug effects
  • Oocytes / metabolism
  • Picrotoxin / pharmacology
  • Protein Conformation / drug effects
  • Protein Subunits
  • Receptors, GABA-B / chemistry*
  • Receptors, GABA-B / genetics
  • Receptors, GABA-B / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Xenopus laevis
  • gamma-Aminobutyric Acid / pharmacology

Substances

  • Fluorescent Dyes
  • GABA type B receptor, subunit 1
  • GABA-B Receptor Agonists
  • GABA-B Receptor Antagonists
  • Protein Subunits
  • Receptors, GABA-B
  • Recombinant Proteins
  • Picrotoxin
  • gamma-Aminobutyric Acid
  • Cysteine