Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter.
Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.
Matsumoto
Takuya
T
Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan.
Kanamoto
Takuji
T
Otsuka
Masato
M
Omote
Hiroshi
H
Moriyama
Yoshinori
Y
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D005786:Gene Expression Regulation; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D027701:Organic Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D013379:Substrate Specificity
D027701:Organic Cation Transport Proteins; C521869:SLC47A1 protein, human; D018698:Glutamic Acid