Photoaffinity-Labelling of the Glycine Receptor of Rat Spinal Cord

Abstract

The irreversible incorporation upon UV illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord was investigated. The specificity of this photoaffinity-labeling reaction for the glycine receptor was demonstrated by the following results: the Kd value (9.7 nM) of the glycine-displaceable irreversible incorporation of [3H]strychnine was similar to the previously reported Kd of [3H]strychnine binding to the glycine receptor; pre-ilumination of the membranes with unlabeled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; the UV light-induced incorporation into the membranes of [3H]strychnine was inhibited by different glycine receptor agonists; other neurotransmitter substances had little or no effect. Also, [3H]strychnine alone was shown to be stable upon illumination with UV light; this suggests that photocrosslinking of [3H]strychnine may require energy transfer from specific groups of its high-affinity receptor binding site. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis a single labeled polypeptide with a relative MW of 48,000 was revealed from spinal cord membranes photoaffinity-labeled with [3H]strychnine. Spinal cord membranes photoaffinity-labeled with the GABA receptor ligand [3H]flunitrazepam gave a single polypeptide with a relative MW of 50,000. Treatment of membranes, labeled with [3H]strychnine, or by endoglycosidase H did not alter the relative MW of the 48,000 labeled polypeptide. Trypsin treatment successively produced major fragments of relative MW of 42,000 and 37,000. Even after extensive treatment with trypsin or chymotrypsin, .gtoreq. 90% of the radioactivity incorporated into the labeled membranes remained membrane-associated. The strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e., probably hydrophobic domain of the 48,000-MW subunit.