Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein toa cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine

Introduction: P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferr ing the multidrug resistance (MDR) phenotype. Methods: In this paper we use d the 1,4-dihydropyridine (I,4-DHP) MDR-reversing agent dexniguldipine (DN) , and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [H-3]-vinblastine transp ort by P-gp. Results: In cytotoxicity assays, 1 mu M DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit wi th an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive. In photoaffi nity labelling studies using the arylazide [H-3]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp wh ilst N-methyl-DN was without effect. However, in photoaffinity labelling st udies in membrane fragments, both DN and N-methyl-DN potently inhibited [H- 3]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane frag ments [H-3]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (K-i 11.2 +/- 3.8 nM), and bo th N-methyl-DN and DN blocked ATP-dependent [H-3]-vinblastine transport int o inside-out vesicles. Thus, in intact cells the permanently charged 1,4-di hydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoa ffinity labelling of P-gp. However, in cell fragments and inside-out vesicl es, N-methyl-DN binds avidly to P-gp and this binding blocks [H-3]-vinblast ine transport. Conclusion: These data are consistent with the hypothesis th at 1,4-DHPs block [H-3]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.