A single β subunit M2 domain residue controls the picrotoxin sensitivity of αβ heteromeric glycine receptor chloride channels

Abstract

This study investigated the residues responsible for the reduced picrotoxin sensitivity of the ab heteromeric glycine receptor relative to the a homomeric receptor. By analogy with structurally related receptors, the b subunit M2 domain residues P278 and F282 were considered the most likely candidates for mediating this effect. These residues align with G254 and T258 of the a subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the a homomeric receptor. Furthermore, the converse F282T mutation in the b subunit increased the picrotoxin sensitivity of the ab heteromeric receptor. The P278G mutation in the b subunit did not affect the picrotoxin sensitivity of the ab heteromer. Thus, a ring of ®ve threonines at the M2 domain depth corresponding to a subunit T258 is speci®cally required for picrotoxin sensitivity. Mutations to a subunit T258 also profoundly in¯uenced the apparent glycine af®nity. A substituted cysteine accessibility analysis revealed that the T258C sidechain increases its pore exposure in the channel open state. This provides further evidence for an allosteric mechanism of picrotoxin inhibition, but renders it unlikely that picrotoxin (as an allosterically acting`competitive' antagonist) binds to this residue.

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

“…Furthermore, other reports have demonstrated little or no usefacilitated block by picrotoxin in glycine and GABA C receptors, respectively (13,21). Thus, picrotoxin might bind at an allosteric site to stabilize a closed or desensitized state of these channels (11,12,27). To resolve the complexity of the picrotoxin interaction with these channels, the existence of multiple binding sites for picrotoxin has been suggested (7,10).…”

“…Several studies have implicated residues in the cytoplasmic aspect of TMII as the picrotoxin binding site (27, 29 -31). Mutations introduced at both the 2Ј and 6Ј positions of TMII confer PTX resistance (15,16,27). Picrotoxin can protect a cysteine engineered into the 2Ј position from irreversible modification by reactive sulfhydryl reagents (29).…”

Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

“…Both picrotoxin and the native ginkgolides have been shown to form interactions with the 6Ј residues in the M2 helices lining the ion channel pore (18 -20, 25, 27, 28). Furthermore, the 2Ј M2 residue, located one helix turn below, has been proposed to be involved in the binding of picrotoxin (25,28) and to be involved in the coordination of the ginkgolides to the heteromeric ␣1␤ GlyR but not to the homomeric GlyR (19,20). In structure-activity relationship studies of ginkgolide analogs, the GlyR antagonist activity of the ginkgolide has been demonstrated to be very dependent on its rigid structure, and modifications of the hydroxyl groups in the molecule have been found to have detrimental effects on its activity (24,29).…”

“…The 6Ј residue in the bottom half of the M2 ␣-helices forming the ion channel of the GlyR have been shown to be involved in binding of native ginkgolides as well as picrotoxin and its two components picrotin and picrotoxinin (19,20,25,27,28). The 6Ј residue is highly conserved through the Cys-loop receptor family, being a Thr or a Ser residue in almost all subunits (Fig.…”

“…Ginkgolides B, C, and J have exhibited preference for heteromeric over homomeric GlyRs in studies of cloned and native receptors (18,19,21). Thus, the compounds display the opposite pharmacological profile compared with that of another natural product, picrotoxin (an equimolar mixture of picrotoxinin and picrotin) (4,25,26). In contrast to picrotoxin, however, the native ginkgolides cannot be claimed to be truly selective against some GlyR subtypes over others, as IC 50…”