• SURFE²R N1

    Easy-to-learn all-in-one device, ideal for teaching and university research
  • SURFE²R N1

    Finally label-free functional assays for transporters available
  • SURFE²R N1

    High signal amplification compared to patch-clamp: transport & binding assays
  • SURFE²R N1

    The only instrument on the market for SSM-based electrophysiology
  • SURFE²R N1

    Turn-key system for efficient transporter protein analysis

1999 - Kinetics of electrogenic transport by the ADP/ATP carrier

Icon N1  SURFE²R-technology (custom-built system) publication in Biophysical Journal (1999)

Authors:
Gropp T., Brustovetsky N., Klingenberg M., Müller V., Fendler K., Bamberg E.

Journal:
Biophysical Journal (1999) 77(2):714-726


Abstract:

The electrogenic transport of ATP and ADP by the mitochondrial ADP/ATP carrier (AAC) was investigated by recording transient currents with two different techniques for performing concentration jump experiments: 1) the fast fluid injection method: AAC-containing proteoliposomes were adsorbed to a solid supported membrane (SSM), and the carrier was activated via ATP or ADP concentration jumps. 2) BLM (black lipid membrane) technique: proteoliposomes were adsorbed to a planar lipid bilayer, while the carrier was activated via the photolysis of caged ATP or caged ADP with a UV laser pulse. Two transport modes of the AAC were investigated, ATPex-0in and ADPex-0in. Liposomes not loaded with nucleotides allowed half-cycles of the ADP/ATP exchange to be studied. Under these conditions the AAC transports ADP and ATP electrogenically. Mg2+ inhibits the nucleotide transport, and the specific inhibitors carboxyatractylate (CAT) and bongkrekate (BKA) prevent the binding of the substrate. The evaluation of the transient currents yielded rate constants of 160 s−1 for ATP and ≥400 s−1 for ADP translocation. The function of the carrier is approximately symmetrical, i.e., the kinetic properties are similar in the inside-out and right-side-out orientations. The assumption from previous investigations, that the deprotonated nucleotides are exclusively transported by the AAC, is supported by further experimental evidence. In addition, caged ATP and caged ADP bind to the carrier with similar affinities as the free nucleotides. An inhibitory effect of anions (200–300 mM) was observed, which can be explained as a competitive effect at the binding site. The results are summarized in a transport model.


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