Functional consequences of NKCC2 splice isoforms: insights from a Xenopus oocyte model

Abstract

The Na+-K+-2Cl− cotransporter NKCC2 is exclusively expressed in the renal thick ascending limb (TAL), where it exists as three main splice isoforms, NKCC2B, NKCC2A, and NKCC2F, with the latter two predominating. NKCC2A is expressed in both medullary and cortical TAL, but NKCC2F localizes to the medullary TAL. The biochemical characteristics of the isoforms have been extensively studied by ion uptake studies in Xenopus oocytes, but the functional consequences of alternative splicing remain unclear. We developed a charge-difference model of an NKCC2-transfected oocyte. The model closely recapitulated existing data from ion-uptake experiments. This allowed the reconciliation of different apparent Km values reported by various groups, which have hitherto either been attributed to species differences or remained unexplained. Instead, simulations showed that apparent Na+ and Cl− dependencies are influenced by the ambient K+ or Rb+ bath concentrations, which differed between experimental protocols. At steady state, under bath conditions similar to the outer medulla, NKCC2F mediated greater Na+ reabsorption than NKCC2A. Furthermore, Na+ reabsorption by the NKCC2F-transfected oocyte was more energy efficient, as quantified by JNKCC/ JPump. Both the increased Na+ reabsorption and the increased efficiency were eroded as osmolarity decreased toward levels observed in the cortical TAL. This supports the hypothesis that the NKCC2F is a medullary specialization of NKCC2 and demonstrates the utility of modeling in analyzing the functional implications of ion uptake data at physiologically relevant steady states.