title: Localization of the 1,4-dihydropyridine drug acceptor of P-glycoprotein to a cytoplasmic domain using a permanently charged derivative N-methyl dexniguldipine.
abstract: BACKGROUND
  P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype.
  
  
  METHODS
  In this paper we used the 1,4-dihydropyridine (1,4-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 [3H]-vinblastine transport by P-gp.
  
  
  RESULTS
  In cytotoxicity assays, 1 microM 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 with an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H]-vinblastine transport.
  
  
  CONCLUSIONS
  These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.
authors.affiliation: CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, UK.
authors.forename: D
authors.initials: D
authors.lastname: Ferry
authors.forename: R
authors.initials: R
authors.lastname: Boer
authors.forename: R
authors.initials: R
authors.lastname: Callaghan
authors.forename: W R
authors.initials: WR
authors.lastname: Ulrich
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000187:drug effects; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism / Q000494:pharmacology*; D001386:Azides / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004095:Dihydropyridines / Q000737:chemistry / Q000494:pharmacology*; D004347:Drug Interactions; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism; D014747:Vinblastine / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D001386:Azides; C094804:B 9209-005; D004095:Dihydropyridines; D014747:Vinblastine; C054074:niguldipine