Identification of a 27-kDa high affinity phenylalkylamine-binding polypeptide as the sigma 1 binding site by photoaffinity labeling and ligand-directed antibodies.

The verapamil-like arylazide (-)-[3H]azidopamil specifically photoaffinity labeled two low molecular mass polypeptides, with apparent molecular masses of 22 and 27 kDa, in the endoplasmic reticulum of guinea pig liver, kidney, adrenal gland, and lung. It was recently shown that the 22-kDa polypeptide binds the anti-ischemic phenylalkylamine (-)-[3H]emopamil and other anti-ischemic drugs with high affinity. We now provide evidence that the photolabeling of the 27-kDa polypeptide is blocked by nanomolar concentrations of sigma ligands [order of potency, haloperidol > pentazocine > 1,3-ditolylguanidine > dextromethorphan > (+)-SKF10,047]. The apparent affinities of these and other drugs closely corresponded to those for 1,3-[3H]ditolylguanidine-labeled sigma binding sites. Based on its high affinity for the (+)-enantiomer [but not the (-)-enantiomer] of SKF10,047 (Ki = 51 nM), pentazocine (Ki = 3 nM), and dextromethorphan (Ki = 30 nM), the (-)-[3H]azidopamil-labeled site on the 27-kDa polypeptide was classified as being of the sigma 1 subtype. Using antiphenylalkylamine antibodies, we developed a novel immunological detection method that allows the rapid and sensitive staining of the photolabeled 27-kDa polypeptide after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We conclude that the phenylalkylamines emopamil and azidopamil represent a novel class of sigma ligands, highly suitable for the further structural characterization of polypeptides carrying sigma 1 binding sites.