Heterogeneity of NH+4 transport in mouse inner medullary collecting duct cells.
Previous studies from our laboratory have demonstrated that NH+4 substitutes for K+ on the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) in rat terminal inner medullary collecting duct cells (tIMCD). To examine other NH+4 transport pathways, a transgenic mouse cell line, mIMCD-3, was employed. 86Rb+ was used as a K+ congener to explore NH+4/Rb+ (K+) competition on the extracellular K+ binding site of the Na(+)-K(+)-2Cl- cotransporter and the Na(+)-K(+)-ATPase. Addition of K+ or NH+4 reduced both bumetanide- and ouabain-sensitive Rb+ uptake. This reduction in Rb+ uptake with NH+4 addition was not due to intracellular pH-mediated changes in transporter activity. K+ and NH+4 are competitive inhibitors on both transporters. On the Na(+)-K(+)-2Cl- cotransporter, the Michaelis constant (Km) for K+ was 4.6 +/- 0.5 mM with an inhibitory constant (Ki) for NH+4 of 2.8 mM. In contrast, on the Na(+)-K(+)-ATPase, the apparent affinity for K+ was greater than for NH+4. To test Na(+)-K(+)-2Cl- cotransport-mediated NH+4 flux, bumetanide-sensitive NH+4/Rb+ exchange was measured. Bumetanide-sensitive Rb+ efflux was greater with extracellular K+ or NH+4 present relative to efflux with extracellular N-methyl-D-glucamine. This demonstrates both K+/Rb+ and NH+4/Rb+ countertransport by the Na(+)-K(+)-2Cl- cotransporter. In conclusion, NH+4 is transported in a bumetanide-sensitive Na(+)-NH+4-Cl- mode, and both NH+4 and Rb+ (K+) are competitive inhibitors for the extracellular K+ binding site. However, the kinetics of Na(+)-K(+)-2Cl(-)-mediated NH+4 transport differ from other K+ transport-mediated NH+4 pathways, such as the Na(+)-K(+)-ATPase.
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D005110:Extracellular Space / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D012413:Rubidium / Q000493:pharmacokinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D027981:Symporters; D000096922:K Cl- Cotransporters
D002034:Bumetanide; D002352:Carrier Proteins; D007477:Ions; D011188:Potassium; D000644:Quaternary Ammonium Compounds; D012413:Rubidium; D000254:Sodium-Potassium-Exchanging ATPase; D027981:Symporters; D000096922:K Cl- Cotransporters