GTP-induced membrane binding and ion channel activity of annexin VI: is annexin VI a GTP biosensor?

Biophys J. 2002 May;82(5):2737-45. doi: 10.1016/S0006-3495(02)75614-2.


Annexin VI (AnxVI) formed ion channels in planar lipid bilayers that were induced by the addition of millimolar guanosine 5'-triphosphate (GTP) at pH 7.4 and that were not accompanied by a penetration of the protein into the membrane hydrophobic region. GTP-influenced interactions of AnxVI with Ca2+/liposomes produced small structural alterations as revealed by circular dichroism and infrared spectroscopies. Guanosine 5'-3-O-(thio)-triphosphate (GTPgammaS) binding to AnxVI, promoted by the photorelease of GTPgammaS from GTPgammaS[1-(4,5-dimethoxy-2-nitrophenyl)-ethyl] (caged-GTPgammaS), affected three to four amino acid residues of AnxVI in the presence of Ca2+/liposomes, while about eight or nine amino acid residues were altered in their absence. This suggested that the nucleotide-binding site overlapped the lipid-binding domain of AnxVI. The binding of the fluorescent GTP analog, 2'-(or 3')-O-(2,4,6-trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) to AnxVI was optimal in the presence of Ca2+/liposomes, with a dissociation constant (K(d)) of 1 microM and stoichiometry of 1. TNP-GTP promoted fluorescence resonance energy transfer from tryptophan residues to the nucleotide. Ion conductance and fluorescence measurements of the C- and N-terminal fragments of AnxVI indicated distinct GTP-binding properties, suggesting that the existence of the GTP-induced ion channel activity of AnxVI is associated with the flexibility of the two halves of the protein. Such structural flexibility could contribute to a molecular mechanism of AnxVI acting as a GTP biosensor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Annexin A6 / isolation & purification
  • Annexin A6 / metabolism*
  • Biosensing Techniques
  • Circular Dichroism
  • Detergents
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / pharmacokinetics*
  • Guanosine Triphosphate / physiology*
  • Humans
  • Ion Channels / chemistry
  • Ion Channels / physiology*
  • Lipid Bilayers / chemistry
  • Membrane Proteins / metabolism*
  • Octoxynol
  • Polyethylene Glycols
  • Protein Conformation
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spectrophotometry, Infrared


  • Annexin A6
  • Detergents
  • Ion Channels
  • Lipid Bilayers
  • Membrane Proteins
  • Recombinant Proteins
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Polyethylene Glycols
  • Guanosine Triphosphate
  • Octoxynol
  • Nonidet P-40
  • GTP-Binding Proteins