Site-directed mutagenesis investigation of coupling properties of metal ion transport by DCT1.
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 reduced the slip currents. These results demonstrate the importance of these regions in coupling of metal ions and protons as well as the possible proximity of I144 and F227 in the folded structure of DCT1.
Nevo
Yaniv
Y
Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. nevoyani@post.tau.ac.il
D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D003035:Cobalt / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D007532:Isoleucine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009865:Oocytes / Q000187:drug effects; D010649:Phenylalanine / Q000235:genetics; D012964:Sodium / Q000494:pharmacology; D014981:Xenopus; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D003035:Cobalt; D007532:Isoleucine; D050505:Mutant Proteins; D010649:Phenylalanine; D012964:Sodium; D000099236:Solute Carrier Family 11, Member 2