The Synthesis and Applications of Isotopically Labelled Compounds 505

~DIOCHEMICALS IN THE STUDY OF RECEPTORS.

H. Glossmann, J. Striessnig, H.-G. Knaus, J. Miiller, G. Zernig, T. Moshammer and A.Grassegger.

Institute o f Biochemical Pharmacology, University o f lnnsbruck, Peter Mayrstr. 1, A-6020 lt,nsbruck; A U S T R I A

All macromolecules which receive a physiological signal and convert it into a cellular response are termed receptors. In pharmacology and toxicology this definition is broader. Here receptors are all macromolecules which interact in a specific manner with a ligand and alter cellular function. A common feature of both physiological and pharmacological receptors is, that the interaction with a ligand or signal is noncatalytic, in many cases reversible, of high affinity and often (with respect to chiral molecules as ligands) enantioselective. Although the physiologist John Newport Langley and the iatrochemist Paul Ehrlich postulated "receptors" nearly a century ago, direct proof their existence was lacking. In pharmacology receptors were for decades mathematical terms, useful - but mysterious. This situation chan~ed dramatically when radioisotopes were

3 35 125 ,732 introduced into receptor research. In general [ H], [ S], [ I] or [ P] are employed on ligands to probe for receptors. Receptors for hydrophilic physiological signals (e.g. hormones, neurotransmitters etc) reside on plasma membranes and their density (e.g. in fmoles/mg protein) is very low. Despite this handicap many receptors have now been isolated, purified, their amino acid sequence deduced from the cloned eDNA, and they are functionally reconstituted in arteficial membranes. In some cases (e.g. enkephalins) the endogeneous, physiological ligands were discovered after the receptors were characterized by radlolabelled drugs. The approach to characterize a receptor with radiolabels will be illustrated by our own work on plasma membrane calcium channels. Calcium antagonists (e.g. 1,4-dihydropyridines, phenylalkylamines, benzothiazepines and dipJ3enyibutylpiperidines) are a diverse group of organic compounds which inhibit transmembrane flow of Z"b • Ca tons m many electrically excitable cells (e.g. heart and smooth muscle and neurons). The very low (riM) effective concentrations, the stereoselectivity and structure-activity-relationships suggested that these compounds interacted in a very specific manner with macromolecules on plasma membranes - i.e. receptors on calcium channels. The first step to characterize these acalcium antagonist receptors was with trltium-labelled 1,4- dihydropvridines, followed by the benzothiazepine [JH]-d-cis-diltiazem and the phenylalkylamines verapamil and (-)-[3H]desmethoxyverapamil. 35S-labelled probes and 1251-labeled derivatives with high very specific activity as well as affinity (K~r-, values in < nM range) were also developed. Several labelled photoaffinity probes ([3H]azidopine, [N-methyl-'~]LU 49888, azido-diltiazem) are extremely useful to identify the drug-receptor carrying polypeptide in solubilized, purified preparations. The purified receptors were reconstituted in artificial planar lipid bilayers and are functional calcium channels. The eDNA of the drug receptor carrying subunit (alpha1) of this L-type calcium channel was cloned and the deduced amino acid sequence shows striking homology to the voltage-dependent sodium channel. Future studies will identify the drug-binding domains within the primary sequence and help to understand drug action as well as channel function at the molecular level. WORK SUPPORTED BY FONDS ZUR FORDERUNG DER WISSENSCHAFTLICItEN FORSCHUNG, DEUTSCHE FORSCItUNGSGEMEINSCffAFT AND BY BUNDESMINISTERIUM FOR WISSENSCHAFT UND FORSCHUNG