Lanthanide/proline cotransport across rabbit renal brush borders

Summary

It has been suggested previously that La³⁺ can replace Na⁺ on various cotransport systems in renal brush border membranes. In the present study, we used rabbit renal brush border membrane vesicles to examine the specificity and kinetics of Ln³⁺/proline cotransport. Experiments were carried out under zero-trans, voltage clamped conditions using a rapid-mix/filtration technique. Initial experiments confirmed that La³⁺ produced the classical overshoot phenomenon. The initial rates of proline uptake relative to Na⁺ were Eu³⁺, Tb³⁺, Nd³⁺, Pr³⁺, Ho³⁺ (3.3)>Na⁺ (1.0)>La³⁺ (0.86) > choline⁺ (0.1). At a saturating salt concentration, uptake saturated with increasing proline concentration: theK _t andJ _max were 0.05mm and 17 pmol mg⁻¹ sec⁻¹ in Na⁺; and 0.28mm and 73 pmol mg⁻¹ sec⁻¹ in Tb³⁺. The higherJ _max in Tb³⁺ indicates that the Tb³⁺-proline loaded carrier is more effective than the Na⁺-proline loaded carrier in overcoming some rate-limiting barriers in the transport process. Na⁺ activated proline uptake with a Hill coefficient of 1.6 and aK _0.5 of 21mm, while Tb³⁺ activated with a Hill coefficient of 0.88 and aK _0.5 of 28mm. The Hill coefficient for Na⁺ suggests two binding sites, whereas the Hill coefficient for Tb³⁺ may indicate negative cooperativity between the trivalent ligands at the binding sites. We conclude that lanthanides are able to substitute for Na⁺ on the brush border proline carrier and that the lanthanides may serve as useful probes for the ligand binding sites.