Effect of phosphonoformic acid, dietary phosphate and the Hyp mutation on kinetically distinct phosphate transport processes in mouse kidney

Abstract

We examined the kinetics of phosphate transport in mouse renal brush-border membrane vesicles under initial rate (6 s), trans zero, voltage clamp conditions. Two kinetically distinct Na +-dependent phosphate transport processes were identified: a high-affinity, low-capacity system ( K m, 0.09 ± 0.02 mM; 539±50 pmol/mg protein per 6 s) and a low-affinity, high-capacity system ( K m, 1.28 ± 0.35 mM; V max, 1677±198 pmol/mg protein per 6 s). The high-affinity system was inhibited competitively by 1 mM phosphonoformic acid (PFA) (apparent K i, 0.31 ± 0.03 mM) and completely abolished by 20 mM PFA; the low-affinity system was insensitive to 1 mM PFA and was inhibited competitively by 20 mM PFA (apparent K i 9.03 ± 1.21 mM). Dietary phosphate deprivation elicited a significant increase in V max of both high- and low-affinity phosphate transport systems whereas the X-linked Hyp mutation caused a 50% decrease in V max of the high-affinity system with no change in the low-affinity system. Phosphate deprivation of Hyp mice elicited a 3.5-fold increase in V max of the high-affinity system. Neither diet nor mutation significantly altered the apparent K m values of either phosphate transport process. We conclude that (1) mouse kidney brush-border membranes have two distinct Na +-dependent phosphate transport systems which differ in affinity and capacity; (2) both processes participate in the adaptive response to dietary phosphate restriction; (3) only the high-affinity system is impaired by the X-linked Hyp mutation.