Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells.
Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP(+)). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH(2)-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (approximately 55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP(+) uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP(+) uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent V(max) at pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent K(m) values were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP(+) uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.
Xia
Li
L
Dept. of Pharmaceutics, University of Washington, Seattle, WA 98195-7610, USA.
Engel
Karen
K
Zhou
Mingyan
M
Wang
Joanne
J
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000241:Adenosine / Q000494:pharmacology; D000818:Animals; D000917:Antibody Formation; D002462:Cell Membrane / Q000737:chemistry*; D002478:Cells, Cultured; D017338:Cladribine / Q000494:pharmacology; D004285:Dogs; D033705:Equilibrative Nucleoside Transport Proteins; D006632:Histamine / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000737:chemistry* / Q000166:cytology; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000276:immunology / Q000378:metabolism*; D014372:Tubercidin / Q000031:analogs & derivatives / Q000494:pharmacology
D033705:Equilibrative Nucleoside Transport Proteins; D009419:Nerve Tissue Proteins; D027701:Organic Cation Transport Proteins; C495988:SLC29A4 protein, human; D017338:Cladribine; C053222:2'-deoxytubercidin; D006632:Histamine; D000241:Adenosine; D014372:Tubercidin; D015655:1-Methyl-4-phenylpyridinium