Site-directed mutagenesis investigation of coupling properties of metal ion transport by DCT1 (original) (raw)

2008, Biochimica et biophysica acta

DCT1 (NRAMP2, DMT1, slc11a2) is a member of the NRAMP family and functions as general metal ion transporter in mammals; defective DCT1 causes anemia. The driving force for metal ion transport is protonmotive force, where protons are transported in the same direction as metal ions. The stoichiometry between metal ion and proton varies under different conditions due to mechanistic proton slip. To better understand this phenomenon, we performed site-directed mutagenesis of DCT1 and analyzed the mutants by measurement of metal ion uptake activity and electrophysiology in Xenopus laevis oocytes. A single reciprocal mutation, I144F, between DCT1 and the homologous yeast transporter Smf1p located in putative transmembrane domain 2 abolished the metal ion transport activity of DCT1, significantly increased the slip currents, and generated sodium slip currents. A double mutation adding F227I in transmembrane domain 4 to I144F in transmembrane domain 2 restored the uptake activity of DCT1 and...