Agonist‐ and antagonist‐induced conformational changes of loop F and their contributions to the ρ1 GABA receptor function

Binding of γ‐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 ρ1 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.