Phosphate (Pi) levels are tightly controlled in humans and elevated Pi in the blood may lead to atherosclerosis and endothelial dysfunction. This is a particular problem in patients with renal failure on dialysis who often suffer from hyperphosphataemia. The current strategies to reduce intestinal Pi uptake in these patients are accompanied by unwanted side effects. Pi transport in the gut is mediated by a member of the Na/Pi cotransporter family, Slc34a2 or NaPi-IIb. Detailed knowledge of how NaPi-IIb recognises and binds Pi may therefore lead to improved blockers of intestinal Pi uptake. We have pursued a comparative approach to identify candidate regions that influence Pi affinity and specificity of NaPi-IIb. To investigate Pi affinity we have taken advantage of two NaPi-IIb isoforms from zebrafish that show very close homology on protein level but a 10-fold difference in Km for Pi. Within the functionally relevant regions of the protein (two stretches of 51 amino acids) there are 13 substitutions between the two isoforms. We have performed site-directed mutagenesis and characterised the mutated transporters in Xenopus oocytes. The aim was to gradually shift the Km for Pi from one isoform towards the other.Preliminary results confirm the validity of our approach. A detailed analysis of the different mutants will be presented. Our hypothesis predicting how Slc34a proteins recognise Pi is based on the crystal structure of a bacterial Pi binding protein. Here, a negatively charged acidic side chain stabilises the hydroxyl group of Pi. If the acidic residue is changed to a basic amino acid the protein no longer binds Pi but sulphate (SO42- in contrast to HPO42-)[1]. A sequence alignment of all known Slc34a isoforms identified two highly conserved acidic residues within the functionally important region of the transporter family. We mutated the relevant amino acids in flounder NaPi-IIb (D181 and E424) to cysteine or histidine with the aim of changing substrate specificity of the mutants from phosphate to sulphate. The mutants were expressed in Xenopus oocytes and assayed for transport of both substrates. Preliminary results revealed that the mutants E424C, D181C/424C and D181H/E424H did not transport neither Pi nor sulphate. The other mutants (D181C, D181H, E424H, D181C/E424H ans D181C/E424H) still mediated Pi induced currents. At present, sulphate transport could not be reliably measured in any of the mutants. To conclude, our comparative approach combined with site-directed mutagenesis and functional analysis may reveal clues how Slc34a transporters interact with Pi.