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

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 (Arg 271 -Lys 276 ) toward the N-terminal end of the homomeric ␣1 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 Thr 6 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.

Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

“…On the basis of a substituted cysteine accessibility analysis, it was recently proposed that picrotoxin can induce a conformational change in the M2-M3 loop that is not produced by glycine (14). We thus hypothesized that picrotoxin may produce a different relationship between ⌬I and ⌬F than was produced by glycine.…”

“…We recently concluded on the basis of a substituted cystine accessibility study that it changes the M2-M3 loop conformation in a manner that cannot be achieved by glycine (14). If so, then picrotoxin might be expected to alter ⌬F in a way that cannot be achieved by altering glycine concentration alone.…”

“…Relatively little attention has been given to the possibility that different agonists and pharmacological modulators may promote different structural conformations in the pore region. However, it has been suggested on the basis of substituted cysteine accessibility studies that the pore blocker, picrotoxin, can change the conformation of the outer GlyR pore region suggesting in turn that it may interact allosterically with agonistinduced conformational changes (14). Our aim is to investigate the conformational changes induced by agonists, antagonists, and allosteric modulators by covalently labeling different residues near the extracellular M2 boundary with the sulfhydrylreactive fluorophores, methanethiosulfonate-rhodamine (MTSR) and tetramethylrhodamine-maleimide (TMRM), and simultaneously measuring current changes and fluorescence changes (15,16).…”

Potentiation and inhibition of glycine receptors by tutin

“…NO: not observed. Hawthorne and Lynch, 2005;Wang et al, 2007), once again show a high complexity. For example, tutin was unable to potentiate the two mutants (Fig.…”

“…For example, it was reported that PTX blocks glycine and GABA A receptors by complex mechanisms ranging from open channel blockers on GABA A Rs to allosteric competitive antagonists on GlyRs (Wang-Tilz et al, 2006). The idea that PTX binds within the channel pore is supported by data showing that the a1 R271C mutant, a residue thought to be lying in the pore, is less sensitive to PTX blockade (Etter et al, 1999;Hawthorne and Lynch, 2005). Other studies, in addition, have suggested that PTX also binds additional sites in the GlyR (Wang et al, 2007), including sites at the pore-associated TM2 residues 258 and 254 (Dash et al, 1991;Zhang et al, 1995;Burzomato et al, 2004).…”