Photoaffinity-labelling of the glycine receptor of rat spinal cord.

The irreversible incorporation upon ultraviolet illumination of the glycine receptor antagonist, [3H]strychnine, into synaptic membrane fractions of rat spinal cord has been investigated. The specificity of this photoaffinity-labelling reaction for the glycine receptor was demonstrated by the following results: (a) 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; (b) pre-illumination of the membranes with unlabelled strychnine led to a corresponding reduction in the number, but not the affinity, of reversible glycine-displaceable [3H]strychnine binding sites; (c) the ultraviolet 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 ultraviolet 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 sulphate/polyacrylamide gel electrophoresis a single labelled polypeptide with a relative molecular mass of 48000 was revealed from spinal cord membranes photoaffinity-labelled with [3H]strychnine. Spinal cord membranes photoaffinity-labelled with the gamma-aminobutyric acid receptor ligand [3H]flunitrazepam, however, gave a single polypeptide with a relative molecular mass of 5- 0000. Treatment of membranes, labelled with [3H]strychnine, by endoglycosidase H did not alter the relative molecular mass of the 48000-Mr labelled polypeptide. Trypsin treatment, on the other hand, successively produced major fragments of relative molecular masses of 42000 and 37000. Also, even after extensive treatment with trypsin or chymotrypsin, greater than or equal to 90% of the radioactivity incorporated into the labelled membranes remained membrane-associated. It is concluded that the strychnine binding site of the glycine receptor is located on a protease-inaccessible, i.e. probably hydrophobic domain of the 48000-Mr subunit.