Identification of Essential Histidine and Cysteine Residues of the H⁺/Organic Cation Antiporter Multidrug and Toxin Extrusion (MATE)

Abstract

Multidrug and toxin extrusion 1 (MATE1) has been isolated as an H ϩ /organic cation antiporter located at the renal brushborder membranes. Previous studies using rat renal brushborder membrane vesicles indicated that cysteine and histidine residues played critical roles in H ϩ /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 substraterecognition sites.

Evaluation of 4′,6-Diamidino-2-phenylindole as a Fluorescent Probe Substrate for Rapid Assays of the Functionality of Human Multidrug and Toxin Extrusion Proteins

“…8). These results suggest the inhibition of both hMATEs by thiol modification, which is consistent with the characteristic of the organic cation/H ϩ antiport system in the kidney (Hori et al, 1987) and indicates that cysteine residues, which have a thiol group, may play an important role in the transporting function (Ohta et al, 2006;Asaka et al, 2007).…”

“…Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H ϩ antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs, such as cimetidine, tetraethylammonium (TEA), N-methylnicotinamide, and metformin in renal tubules Tsuda et al, 2007). Two human MATEs, hMATE1 and hMATE2, were originally identified in 2005 by Otsuka et al (2005).…”

Identification of multidrug and toxin extrusion (MATE1 and MATE2-K) variants with complete loss of transport activity

“…These findings suggest that reduced protein expression levels in the plasma membrane can account for the decrease in transport activity of MATE1 with G64D and D328A. Previously, we indicated that Cys63 of MATE1 plays an important role in substrate binding, 22 and Cys63 is the neighboring amino-acid residue of Gly64. Thus, regarding G64D, the change from the small side chain (Gly) to the bulkier and polar side chain (Asp) may inhibit the substrate binding and decrease transport activities, in addition to reduced protein expression levels in the plasma membrane.…”

“…Cell surface biotinylation was performed according to our earlier methods 22 with some modifications. HEK293 cells were grown on poly-D-lysine-coated 12-well plates and transfected with MATE1 or MATE2-K cDNA plasmids (50 ng for MATE1 and 200 ng for MATE2-K).…”

Twelve Transmembrane Helices Form the Functional Core of Mammalian MATE1 (Multidrug and Toxin Extruder 1) Protein

“…In fact, the histidine-specific reagent, diethyl pyrocarbonate (DEPC) inhibited activity of the OC/H ϩ exchanger of rat renal brush border membrane vesicles (53), although the presence of TEA in excess did not protect the transporter from DEPC inhibition. Asaka et al (54) individually replaced each of the seven conserved histidine residues in rMate1 with glutamine residues and found that only the H385Q mutation resulted in a change (50% decrease) in TEA transport. They also found that DEPC inhibition of rMate1-mediated TEA transport is not eliminated by coexposure of DEPC with excess TEA, leading to the suggestion that His-385 is not part of the TEA binding site but, instead, may be part of the H ϩ interaction site (54).…”