Vol. 58, Issue 1, 11-17, July 2000

Subunit-Specific Action of an Anticonvulsant Thiobutyrolactone on Recombinant Glycine Receptors Involves a Residue in the M2 Membrane-Spanning Region

Joe Henry Steinbach, John Bracamontes, Li Yu, Pengnian Zhang, and Douglas F. Covey

Departments of Anesthesiology (J.H.S., J.B., L.Y., P.Z.) and Molecular Biology & Pharmacology (D.F.C.), Washington University School of Medicine, St. Louis, Missouri

The anticonvulsant -ethyl, -methyl--thiobutyrolactone (EMTBL) potentiates the response to a submaximal concentration of glycine produced by receptors composed of human glycine 1-subunits but reduces the response of receptors composed of rat glycine 3-subunits. Both the potentiating and blocking actions of EMTBL are reduced by higher concentrations of glycine. The subunit specificity of EMTBL block is conferred by a residue in the second membrane-spanning region (M2), which is alanine in the 3-subunit (A254) and glycine in the 1-subunit. The mutation A254G in the 3-subunit removes blocking by EMTBL and reveals potentiation. Picrotin, a picrotoxinin analog, blocks responses of receptors composed of either 1 or 3-subunits. Blocking of 3 receptors by picrotin is reduced in the presence of EMTBL, indicating that the mechanisms interact at some point, but the mutation 3 A254G does not remove block by picrotin. However, mutation of a nearby residue 3 T258F does remove block by picrotin, picrotoxinin and EMTBL. These observations suggest that EMTBL and picrotin act on glycine 3 receptors to produce block by an allosteric mechanism that involves overlapping sets of residues in the M2 region. Coexpression of the 3-subunit with the -subunit of the glycine receptor also removes block by EMTBL and reveals potentiation, suggesting that receptors containing either 3 or 1 glycine receptor subunits are potentiated in the adult brain.