Agonist- and antagonist-induced conformational changes of loop F and their contributions to the rho1 GABA receptor function.
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.
Zhang
Jianliang
J
Division of Neurobiology, Barrow Neurological Institute, St Joseph's Hospital and Medical Center, Phoenix, AZ 85013, USA.
Xue
Fenqin
F
Chang
Yongchang
Y
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes; D058786:GABA-B Receptor Agonists; D058788:GABA-B Receptor Antagonists; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010852:Picrotoxin / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D021122:Protein Subunits; D018080:Receptors, GABA-B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D003545:Cysteine; D005456:Fluorescent Dyes; D058786:GABA-B Receptor Agonists; D058788:GABA-B Receptor Antagonists; D010852:Picrotoxin; D021122:Protein Subunits; D018080:Receptors, GABA-B; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid