Characterization of (iodine-125)omega-conotoxin binding to brain nitrogen calcium channels and (-)(hydrogen-3)desmethoxyverapamil binding to novel calcium channels in osteoblast-like osteosarcoma cells.

摘要

This dissertation provides molecular evidence for a diversity of Ca{dollar}sp{lcub}2+{rcub}{dollar} channels in neuronal and non-neuronal tissues. First, I demonstrated specific, reversible, saturable binding sites for omega({dollar}sp{lcub}125{rcub}{dollar}I) conotoxin GVIA (omega({dollar}sp{lcub}125{rcub}{dollar}I) CTX) in rat brain and rabbit sympathetic ganglion. Omega({dollar}sp{lcub}125{rcub}{dollar}I) CTX binding has a unique pharmacology, ion selectivity, and anatomical distribution in rat brain. Omega({dollar}sp{lcub}125{rcub}{dollar}I) CTX binding was solubilized, retaining an appropriate pharmacology and ion selectivity. Omega({dollar}sp{lcub}125{rcub}{dollar}I) CTX binding may be associated with a Ca{dollar}sp{lcub}2+{rcub}{dollar} channel because the K{dollar}sb{lcub}rm D{rcub}{dollar} of omega({dollar}sp{lcub}125{rcub}{dollar}I) CTX is similar to the IC{dollar}sb{lcub}50{rcub}{dollar} of inhibition of depolarization-induced {dollar}sp{lcub}45{rcub}{dollar}Ca{dollar}sp{lcub}2+{rcub}{dollar} flux into rat brain synaptosomes.; Specific ({dollar}-{dollar})({dollar}sp3{dollar}H) desmethoxyverapamil (({dollar}-{dollar})({dollar}sp3{dollar}H) DMV) binding sites were demonstrated on osteoblast-like osteosarcoma cell membranes. Osteosarcoma cells share many characteristics with electrically nonexcitable osteoblasts. ({dollar}-{dollar})({dollar}sp3{dollar}H) DMV binding is stereoselective for the isomers of DMV and inhibited by other phenylalkylamines, although binding is not sensitive to dihydropyridine derivatives. The pharmacology of the ({dollar}-{dollar})({dollar}sp3{dollar}H) DMV is thus different than any previously described phenylalkylamine binding site. The relationship of the ({dollar}-{dollar})({dollar}sp3{dollar}H) DMV binding sites to osteoblast Ca{dollar}sp{lcub}2+{rcub}{dollar} channels is suggested, in part, by the influence of Ca{dollar}sp{lcub}2+{rcub}{dollar} and other divalent cations upon binding. The relationship of the ({dollar}-{dollar})({dollar}sp3{dollar}H) DMV binding sites to osteoblast Ca{dollar}sp{lcub}2+{rcub}{dollar} channels is further suggested by the demonstration of cation conductance in membrane patches from osteosarcoma cells. This conductance is sensitive to phenylalkylamine drugs but not to dihydropyridines, resembling the pharmacology of the ({dollar}-{dollar})({dollar}sp3{dollar}H) DMV binding sites. This phenylalkylamine-sensitive putative Ca{dollar}sp{lcub}2+{rcub}{dollar} in osteoblasts may play a role in the signal transduction of parathyroid hormone.