Detailed characterization of cysteine-less P-glycoprotein reveals subtle pharmacological differences in function from wild-type protein

Br J Pharmacol. 2001 Dec;134(8):1609-18. doi: 10.1038/sj.bjp.0704400.


1. Subtle alterations in the coupling of drug binding to nucleotide hydrolysis were observed following mutation of all seven endogenous cysteine residues to serines in the human multidrug resistance transporter, P-glycoprotein. Wild-type (wt) and the mutant (cys-less) forms of P-gp were expressed in Trichoplusia ni (High Five) cells and purified by metal affinity chromatography in order to undertake functional studies. 2. No significant differences were observed in substrate ([(3)H]-azidopine) binding to wt or cys-less P-gp. Furthermore, neither the transported substrate vinblastine, nor the modulator nicardipine, differed in their respective potencies to displace [(3)H]-azidopine from the wt or cys-less P-gp. These results suggest that respective binding sites for these drugs were unaffected by the introduced cysteine to serine substitutions. 3. The Michaelis-Menten characteristics of basal ATP hydrolysis of the two isoforms of P-gp were identical. The maximal ATPase activity in the presence of vinblastine was marginally reduced whilst the K(m) was unchanged in cys-less P-gp compared to control. However, cys-less P-gp displayed lower overall maximal ATPase activity (62%), a decreased K(m) and a lower degree of stimulation (76%) in the presence of the modulator nicardipine. 4. Therefore, the serine to cysteine mutations in P-gp may suggest that vinblastine and nicardipine transduce their effects on ATP hydrolysis through distinct conformational pathways. The wt and cys-less P-gp isoforms display similarity in their fundamental kinetic properties thereby validating the use of cys-less P-gp as a template for future cysteine-directed structure/function analysis.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / chemistry*
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / genetics
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / physiology*
  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Animals
  • Azides / metabolism
  • Baculoviridae / genetics
  • Binding Sites
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Cross-Linking Reagents / chemistry
  • Cysteine / genetics*
  • Dihydropyridines / metabolism
  • Dose-Response Relationship, Drug
  • Drug Resistance, Multiple
  • Humans
  • Inhibitory Concentration 50
  • Kinetics
  • Mutagenesis
  • Nicardipine / pharmacology
  • Photoaffinity Labels / metabolism
  • Serine / genetics
  • Spodoptera / virology
  • Vinblastine / pharmacology


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Azides
  • Cross-Linking Reagents
  • Dihydropyridines
  • Photoaffinity Labels
  • Serine
  • Vinblastine
  • azidopine
  • Adenosine Triphosphate
  • Nicardipine
  • Adenosine Triphosphatases
  • Cysteine