Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers.

Abstract

A radioactive photoactive dihydropyridine calcium channel blocker, [3H]azidopine, was used to photoaffinity label plasma membranes of multidrug-resistant Chinese hamster lung cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled 150-180-kDa doublet in the membranes from drug-resistant but not from the drug-sensitive parental (DC-3F) cells. A similar radiolabeled doublet was barely detected in a drug-sensitive partial revertant (DC-3F/ADX-U) cell line. The 150-180-kDa doublet exhibited a specific half-maximal saturable photolabeling at 1.07 X 10(-7) M [3H]azidopine. The dihydropyridine binding specificity was established by competitive blocking of specific photolabeling with nonradioactive azidopine as well as with nonphotoactive calcium channel blockers nimodipine, nitrendipine, and nifedipine. In addition, [3H]azidopine photolabeling was blocked by verapamil and diltiazem but was stimulated by excess prenylamine and bepridil suggesting a cross-specificity for up to four different classes of calcium channel blockers. The 150-180-kDa calcium channel blocker acceptor co-electrophoresed exactly with the 150-180-kDa surface membrane glycoprotein (gp150-180 or P-glycoprotein) Vinca alkaloid acceptor from multidrug-resistant cells and was immunoprecipitated by polyclonal antibody recognizing gp150-180. [3H]Azidopine photolabeling of the 150-180-kDa component in the presence of excess vinblastine was reduced over 90%, confirming the identity or close relationship of the calcium channel blocker acceptor and the gp150-180 Vinca alkaloid acceptor. The [3H]azidopine photolabeling of gp150-180 also was reduced by excess actinomycin D, adriamycin, or colchicine, demonstrating a broad gp150-180 drug recognition capacity. The ability of gp150-180 to recognize multiple natural product cytotoxic drugs as well as calcium channel blockers suggests a direct function for gp150-180 in the multidrug resistance phenomenon and a role in the circumvention of that resistance by calcium channel blockers.