The coupling mechanism of P-glycoprotein involves residue L339 in the sixth membrane spanning segment

Alice Rothnie, Janet Storm, Roisin McMahon, Andrew Taylor, Ian D. Kerr, Richard Callaghan

Research output: Contribution to journalArticlepeer-review


The transmembrane (TM) domains in P-glycoprotein (P-gp) contain the drug binding sites and undergo conformational changes driven by nucleotide catalysis to effect translocation. However, our understanding of exactly which regions are involved in such events remains unclear. A site-directed labelling approach was used to attach thiol-reactive probes to cysteines introduced into transmembrane segment 6 (TM6) in order to perturb function and infer involvement of specific residues in drug binding and/or interdomain communication. Covalent attachment of coumarin-maleimide at residue 339C within TM6 resulted in impaired ATP hydrolysis by P-gp. The nature of the effect was to reduce the characteristic modulation of basal activity caused by transported substrates, modulators and the potent inhibitor XR9576. Photoaffinity labelling of P-gp with [(3)H]-azidopine indicated that residue 339C does not alter drug binding per se. However, covalent modification of this residue appears to prevent conformational changes that lead to drug stimulation of ATP hydrolysis.
Original languageEnglish
Pages (from-to)3984-3990
Number of pages7
JournalFEBS Letters
Issue number18
Early online date25 Jun 2005
Publication statusPublished - 18 Jul 2005


  • denosine triphosphatases
  • adenosine triphosphate
  • animals
  • baculoviridae
  • binding sites
  • catalysis
  • cell line
  • cell membrane
  • Coumarins
  • cysteine
  • hydrolysis
  • Insects
  • kKinetics
  • linear models
  • mutagenesis, site-directed
  • nicardipine
  • p-glycoprotein
  • Paclitaxel
  • protein binding
  • protein conformation
  • protein isoforms
  • protein structure, tertiary
  • protein transport
  • quinolines
  • recombinant proteins
  • sulfhydryl compounds
  • time factors
  • vinblastine


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