Poster Presentation 2014 International Biophysics Congress

Transporters of the multidrug resistance: Reconstitution into proteoliposomes and biophysical characterisation (#324)

Patrick Seelheim 1 , Hans-Joachim Galla 1
  1. University of Muenster, Muenster, Germany

Human ABC transporters are involved in a multitude of physiological processes including multi-drug resistance. Despite their medical relevance, little is known about the mechanism of ATP hydrolysis and substrate translocation. We investigated the kinetics of ATP hydrolysis and substrate translocation for the human multi-drug resistance related protein 3 (hMRP3, ABCC3) with the focus on the cooperative interactions between the two ATP hydrolysing nucleotide binding domains (NBDs) and the two substrate translocating transmembrane domains (TMDs) of the functional protein in reconstituted large unilamellar vesicles (LUVs).

Upon stimulation with transport substrates, ATPase activity of hMRP3 increased from a basal level by a factor of three showing positive cooperativity with a Hill coefficient of n   =   2. While we found different affinity constants for all tested substrates, the Hill coefficient and vmax seem to be substrate independent. This supports the idea that substrate-induced cooperativity of the two NBDs in ABCC3 is mediated by the TMDs and not by direct interaction between substrate and NBDs.

Further, we investigated the ATP driven uptake of substrates into the lumen of hMRP3-containing LUVs and found substrate translocation to be a cooperative process again with the same kM value that has been found for the APT activity and a Hill coefficient of n   ≈   2 for all tested substrates. This leads us to the assumption that the two TMDs of ABCC3 act in a highly synchronized manner during substrate translocation.

  1. [1] Seelheim P, Wüllner A and Galla HJ; Substrate translocation and stimulated ATP hydrolysis of human ABC transporter MRP3 show positive cooperativity and are half-coupled; Biophys Chem; 171, 31-37 (2013)