Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells

Abstract

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.

Fig. 1

Characterization of a polyclonal anti-plasma membrane monoamine transporter (PMAT) antibody. A: proposed membrane topology of PMAT. Antibody was developed toward the NH₂-terminal 66 amino acid residues of PMAT (shown in dark). B: Western blot analysis of PMAT protein expressed in Madin-Darby canine kidney (MDCK) cells using the polyclonal antibody (1:1,600 dilution). C: MDCK cells transfected with empty vector (pcDNA3) were permeabilized and stained with anti-PMAT antibody. D: MDCK cells transfected with PMAT were permeabilized and stained with the anti-PMAT antibody. E: MDCK cells transfected with PMAT were permeabilized and stained with prebleed serum. F: MDCK cells transfected with PMAT were stained with anti-PMAT antibody without permeabilization. Antibody used in cell staining was at 1:200 dilution. Permeabilization was carried out with 0.2% Triton X-100. Cell nuclei were counterstained with TO-PRO-3.

Fig. 2

Western blot analysis of PMAT expression in human kidney. Immunoblotting was carried out with the anti-PMAT antibody at 1:1,000 dilution. The antiserum for PMAT was either preabsorbed (Preabs.) with (+) or without (−) the purified glutathione S-transferase (GST)-PMAT NH₂-terminal fusion protein. The same blots were stripped and blotted with an antibody against an internal standard, GAPDH.

Fig. 3

Subcellular localization of PMAT or YFP-PMAT in polarized MDCK cells. The cells were cultured on Corning Costar Transwell filters for 10–12 days to allow cell differentiation. The filters were then sliced and loaded on a coverglass slide. MDCK cells stably expressing PMAT were stained with anti-PMAT Ab (1:200) (A). Alternatively, MDCK cells expressing YFP-PMAT (B) and YFP alone (C) were visualized directly with a confocal fluorescence microscope. Cells were imaged in the x-y plane (top). The lines on these images show the z plane for vertical scanning. Bottom: corresponding vertical images.

Fig. 4

Effect of pH on PMAT-mediated [³H]MPP⁺ uptake. Vector-transfected cells (open bar) and PMAT-transfected cells (solid bar) were incubated with 1 μM [³H]MPP⁺ at pH 6.6, 7.4, and 8.2 for 1 min at 37°C. Each bar represents means (SD); n = 3.

Fig. 5

Effect of pH on PMAT-mediated [³H]MPP⁺ uptake kinetics. A: PMAT-transfected and vector-transfected cells were incubated with varying concentrations of MPP⁺ for 1 min at 37°C under pH 6.6 or pH 7.4. The PMAT-specific uptake was calculated by subtracting the transport activity in control cells. B: Eadie-Hofstee plots under pH 6.6 and pH 7.4.

Fig. 6

Effect of carbonyl cyanide p-trifluormethoxyphenylhydrazone (FCCP) on PMAT-mediated MPP⁺ uptake. Vector and PMAT-transfected cells were incubated at 37°C with 1 μM [³H]MPP⁺ for 1 min in the presence of valinomycin (Valino), FCCP or Valino and FCCP under pH 6.6 (A) or pH 7.4 (B). Each bar represents the means (SD); n = 3.

Fig. 7

Effect of various nucleoside analogs on PMAT-mediated organic cation uptake. Vector-transfected (open bars) and PMAT-transfected cells (solid bars) were incubated at 37°C with 1 μM [³H]MPP⁺ for 1 min (A) or 1μM [³H]histamine for 5 min (B) in the absence (control) or presence of various nucleoside analogs at 1 mM. Each bar represents the means (SD); n = 3. Ade, adenosine; AraA, 9-β-D-arabinofuranosyladenine; AraC, 9-β-D-arabinofuranosylcytosine; AZT, azidothymidine; CdA, 2-chloro-2′-deoxyadenosine; ddC, 2′,3′-dideoxycytidine; ddI, 2′,3′-dideoxyinosine; ddT, 2′,3′-dideoxythymidine; dTub, 2′-deoxytubercidin (7-deaza-2′-deoxyadenosine). *Significantly different from control, P < 0.01.