Magnitude of a Conformational Change in the Glycine Receptor β1-β2 Loop Is Correlated with Agonist Efficacy

Joseph W. Lynch,Stephan A. Pless

doi:10.1074/jbc.m109.048405

Abstract

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.

Highlights

National Health and Medical Research Council of Australia (NHMRC). □S The on-line version of this article contains supplemental Fig. S1. 1 Supported by an International Postgraduate Research Scholarship from the
This study suggested that agonist efficacy depends on the ability of the agonist to convert the inert agonist-bound receptor to the pre-open flip state
To facilitate comparison of agonist-induced responses, the maximum currents (⌬Imax) and maximum fluorescence changes (⌬Fmax) elicited by ␤-alanine and taurine at all labeled residues investigated here were normalized to those elicited by glycine via the relations, RI ϭ (⌬Imax(␤-Ala or tau))/(⌬Imax(Gly)) and RF ϭ (⌬Fmax(␤-Ala or tau))/ (⌬Fmax(Gly)), respectively

Summary

EXPERIMENTAL PROCEDURES

Chemicals—Glycine, taurine, and ␤-alanine (all Sigma Aldrich) were dissolved in water and stored at 4 °C. Oocytes were incubated for 3–5 days at 18 °C in a solution containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, 0.6 mM theophylline, 2.5 mM pyruvic acid, 50 ␮g/ml gentamycin (Cambrex Corporation, East Rutherford, NJ), pH 7.4. Prior to recording oocytes were transferred into ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES, pH 7.4) containing 5–20 ␮M dye for 30 s (for MTSlinked dyes) or 45 min (for AF546). All cells were voltage-clamped at Ϫ40 mV and current recordings were made with a Gene Clamp 500B amplifier (Axon Instruments, Union City, CA). Fluorescence and current traces were acquired at 200 Hz with a Digidata 1322A interface and digitally filtered at 2 Hz with an eight-pole Bessel filter for analysis and display (Axon Instruments, Union City, CA). The molecular model was generated with PyMOL 0.99 (DeLano Scientific LLC, San Francisco, CA)

RESULTS

Taurine nH

DISCUSSION