HETEROGENEITY OF NH4+ TRANSPORT MOUSE INNER MEDULLARY COLLECTING DUCTCELLS

Previous studies from our laboratory have demonstrated that NH4+ subst itutes for K+ on the Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) in rat terminal inner medullary collecting duct cells (tIMCD). To examin e other NH4+ transport pathways, a transgenic mouse cell line, mIMCD-3 , was employed. Rb-86(+) was used as a K+ congener to explore NH4+/Rb (K+) competition on the extracellular K+ binding site of the Na+-K+-2 Cl(-) cotransporter and the Na+-K+-ATPase. Addition of K+ or NH4+ redu ced both bumetanide- and ouabain-sensitive Rb+ uptake. This reduction in Rb+ uptake with NH+4 addition was not due to intracellular pH-media ted changes in transporter activity. K+ and NH4+ are competitive inhib itors on both transporters. On the Na+-K+-2Cl(-) cotransporter, the Mi chaelis constant (K-m) for K+ was 4.6 +/- 0.5 mM with an inhibitory co nstant (K-i) for NH4+ of 2.8 mM. In contrast, on the Na+-K+-ATPase, th e apparent affinity for K+ was greater than for NH4+. To test Na+-K+-2 Cl(-) cotransport-mediated NH4+ flux, bumetanide-sensitive NH4+/Rb+ ex change was measured. Bumetanide-sensitive Rb+ efflux was greater with extracellular K+ or NH4+ present relative to efflux with extracellular N-methyl-D-glucamine. This demonstrates both K+/Rb+ and NH4+/Rb+ coun tertransport by the Na+-K+-2Cl(-) cotransporter. In conclusion, NH4+ i s transported in a bumetanide-sensitive Na+-NH4+-Cl- mode, and both NH 4+ and Rb+ (K+) are competitive inhibitors for the extracellular K+ bi nding site. However, the kinetics of Na+-K+-2Cl(-)-mediated NH: transp ort differ from other K+ transport-mediated NH4+ pathways, such as the Na+-K+-ATPase.