TWO REPETITIVE REGIONS ARE ESSENTIAL FOR THE FUNCTION OF THE RENAL TYPE IIa Na+/Pi COTRANSPORTER

Katja Köhler, Ian C. Forster, Georg Lambert, Jürg Biber and Heini Murer
Physiology Institute, University of Zurich, Switzerland

The type IIa Na+/Pi cotransporter (NaPi-IIa) is the major mediator of Pi reabsorption at the brush border membrane of the renal proximal tubule and it therefore plays an important role in Pi homeostasis. The kinetics of NaPi-IIa have been characterized, however little is known about the structural domains responsible for its function. The rat type IIa isoform is a 637 amino acid protein comprising 8 transmembrane domains. Sequence comparisons between the N- and C-terminal halves of the protein revealed two highly homologous regions: the 1^st intracellular (ICL-1) and the 3^rd extracellular loop (ECL-3). This suggests that they may subserve a complementary functional role.
To identify functionally important sites, we applied the substituted cysteine accessibility method (SCAM) to ECL-3 by exchanging selected residues S437 through L465 each to a cysteine. Mutants were expressed in X. laevis oocytes and transport function and kinetics were studied by two electrode voltage clamp. Accessibility of novel cysteines by MTS reagents was determined by comparing transport function before and after application. Suppression of transport function after modification of cysteines in the region from T451 to S460 indicated that this part is functionally important. Moreover, the periodic MTSEA accessibility pattern suggested the presence of an a- helical motif. To further investigate the functional impact of this region, the kinetic characteristics of the mutants were studied. Mutants in ECL-3 showed no change in apparent substrate affinity, but altered voltage and pH dependency, suggesting that part of ECL-3 is located within the electric field and may contain a proton binding site.
SCAM was also applied to ICL-1. Amino acids in ICL-1 that are homologous to those mutated in ECL-3 were exchanged with a cysteine. Consistent with hydropathy predictions, mutants in ICL-1 were less accessible by MTSEA than those in ECL-3. None of the mutants in ICL-1 showed an altered pH dependency, but one mutant (N199C) displayed a significantly reduced apparent affinity for Pi and Na+. To further characterize the role of this residue, we made conservative and non-conservative substitutions at this site. Changing this residue to a T or A dramatically increased the Km for Pi, whereas other substitutions led to expressed, but non-functional transporters. The results suggest that site N199 is a critical residue for substrate binding.
These findings indicate that the two highly conserved regions in the N- and C-terminal halves of the NaPi-IIa protein contribute to essential functional properties of the protein: ECL-3 confers kinetic properties, whereas ICL-1 confers the substrate affinities.