Identification of essential histidine and cysteine residues of the H+/organic cation antiporter multidrug and toxin extrusion (MATE).
Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H(+)/organic cation antiporter located at the renal brush-border membranes. Previous studies using rat renal brush-border membrane vesicles indicated that cysteine and histidine residues played critical roles in H(+)/organic cation antiport activity. In the present study, essential histidine and cysteine residues of MATE1 family were elucidated. When 7 histidine and 12 cysteine residues of rat (r)MATE1 conserved among species were mutated, substitution of His-385, Cys-62, and Cys-126 led to a significant loss of tetraethylammonium (TEA) transport activity. Cell surface biotinylation and immunofluorescence analyses with confocal microscopy indicated that rMATE1 mutant proteins were localized at plasma membranes. Mutation of the corresponding residues in human (h)MATE1 and hMATE2-K also diminished the transport activity. The transport of TEA via rMATE1 was inhibited by the sulfhydryl reagent p-chloromercuribenzenesulfonate (PCMBS) and the histidine residue modifier diethyl pyrocarbonate (DEPC) in a concentration-dependent manner. The PCMBS-caused inhibition of the transport via rMATE1 was protected by an excess of various organic cations such as TEA, suggesting that cysteine residues act as substrate-binding sites. In the case of DEPC, no such protective effects were observed. These results suggest that histidine and cysteine residues are required for MATE1 to function and that cysteine residues may serve as substrate-recognition sites.
Asaka
Jun-ichi
J
Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. inui@kuhp.kyoto-u.ac.jp.
Terada
Tomohiro
T
Tsuda
Masahiro
M
Katsura
Toshiya
T
Inui
Ken-ichi
K
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D017920:Antiporters / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D019789:Tetraethylammonium / Q000378:metabolism; D014118:Toxins, Biological / Q000378:metabolism
D017920:Antiporters; D027701:Organic Cation Transport Proteins; C515734:Slc47a1 protein, rat; D014118:Toxins, Biological; D006639:Histidine; D002731:4-Chloromercuribenzenesulfonate; D019789:Tetraethylammonium; D003545:Cysteine; D004047:Diethyl Pyrocarbonate