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 αβ heteromeric glycine receptor relative to the α homomeric receptor. By analogy with structurally related receptors, the β 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 α subunit. The T258A, T258C and T258F mutations dramatically reduced the picrotoxin sensitivity of the α homomeric receptor. Furthermore, the converse F282T mutation in the β subunit increased the picrotoxin sensitivity of the αβ heteromeric receptor. The P278G mutation in the β subunit did not affect the picrotoxin sensitivity of the αβ heteromer. Thus, a ring of five threonines at the M2 domain depth corresponding to α subunit T258 is specifically required for picrotoxin sensitivity. Mutations to α subunit T258 also profoundly influenced the apparent glycine affinity. 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.