A Picrotoxin-specific Conformational Change in the Glycine Receptor M2–M3 Loop

Rebecca Hawthorne,Joseph W Lynch

doi:10.1074/jbc.m506645200

Abstract

The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.

Highlights

It is well established that the extramembranous loop joining M2 and M3 is crucial for transmitting the agonist-induced conformational change to the M2 domain activation gate [5,6,7,8]
It would address the hypothesis that the M2–M3 loop conformation can be modified in different ways by different ligands
PTX Interactions with the M2–M3 Loop—The peak current magnitudes and glycine EC50 values for all mutant glycine receptor chloride channel (GlyR) examined in this study have previously been published [9]

Summary

A Picrotoxin-specific Conformational Change in the Glycine Receptor M2–M3 Loop*

It is well established that the extramembranous loop joining M2 and M3 is crucial for transmitting the agonist-induced conformational change to the M2 domain activation gate [5,6,7,8] Consistent with this role, a substituted cystine accessibility study on the ␣1 GlyR showed that the surface accessibility of 6 contiguous cysteine-substituted residues in this loop (Arg271 to Lys276) was increased in the open state [9]. Data obtained using a variety of techniques including single channel kinetic and conductance analysis [10, 19], macroscopic current kinetic analysis [20], interactions between picrotoxin analogs [21], site-directed mutagenesis [12, 22], and combined fluorescence labeling and electrophysiology [23] together form a strong case that PTX allosterically inhibits anionic LGICs. PTX may be a useful modulator for the present investigation as mutations in the M2–M3 loop have profound effects on its mode of action [12]

MATERIALS AND METHODS

RESULTS

DISCUSSION