Inorganic phosphate (Pi) is an essential nutrient involved in a variety of cellular and extra-cellular processes. The vectorial transport of anionic Pi across the lumen of epithelia is mediated by members of two genetically unrelated protein families, SLC34 and SLC20, which catalyse transport in a Na-dependent manner (1). At the lumen of renal proximal tubule epithelia, the electrogenic SCL34A1 (NaPi-IIa) and electroneutral SLC34A3 (NaPi-IIc) are the main players responsible for reabsorption of Pi from the urine. They cotransport divalent Pi, whereas the electrogenic SLC20A2 (PiT-2) cotransports monovalent Pi. In the small intestine SLC34A2 (NaPi-IIb) is at least partly responsible for dietary absorption of divalent Pi. The membrane protein abundance determines net Pi transport and their membrane targeting and retrieval is under the control of Pi levels, together with hormonal and various circulating factors. Understanding the transport mechanism at the molecular level and identifying function-specific amino acid residues is essential for defining the physiological role of these transporters and for the development of pharmaceutical agents to target them specifically. Both SLC20 and SLC34 proteins have a predicted 12 transmembrane domain topology with inverted repeat architectures. As no 3-D structural models are available for these proteins and their bacterial homologs show no similarity to transporters whose architectures are currently identified, we must rely on indirect experimental approaches to gain structure-function information. Our studies have largely focussed on over-expression of the proteins in Xenopus oocytes. By performing real-time functional assays (electrophysiology and fluorometry) in combination with site-directed mutagenesis for substituted cysteine scanning accessibility (SCAM) assays, we have identified functionally important sites, established topological features and have begun to elucidate the dynamics of protein conformation changes during the transport cycle, under physiological conditions. Using SCAM, in which novel cysteines are substituted at sites predicted to be functionally sensitive, labelled with methanethiosulfonate reagents and their accessibility from the aqueous milieu quantified, we could confirm and refine topological predictions obtained from bioinformatics. For example, the intra- and extracellular accessibility of two short reentrant regions, previously hypothesised to form the transport pathway of NaPi-IIa/b, was established. Recently, using a crosslinking strategy, we obtained compelling evidence that for NaPi-IIb, these regions physically associate (2). The marked difference in electrogenic activity for the two renal SLC34 isoforms, which otherwise show >90% sequence identity in the putative transmembrane spanning regions, prompted us to investigate the underlying molecular determinants. By means of sequence comparison, we identified and experimentally confirmed the location of critical residues that confer electrogenicity to NaPi-IIa/b and moreover, define their transport stoichiometries (3Na+:Pi for NaPi-IIa /b and 2Na+:Pi for NaPi-IIc) and Pi concentrating capacity (3). Furthermore, we established experimentally that 3 Na+ ions interact with the SLC34 proteins, but for the electroneutral NaPi-IIc, the first Na+ ion to bind is not cotransported, thereby providing evidence that transport and binding stoichiometries are not necessarily equivalent (4). Recently we have applied voltage clamp fluorometry (VCF) to investigate conformational changes during the transport cycle. Novel cysteine residues at functionally important sites were labelled with fluorophores and real-time changes in fluorescence emission intensity induced by changes in membrane potential or substrate activity were detected. These indicate an altered microenvironment of the fluorophore that is sensitive to the protein conformation. Using VCF, we have established the order of cation interaction (4,5) and we have obtained evidence of complementary movements of the protein when substrates bind and debind (6). Taken together, these and other studies have allowed us to generate a kinetic model for SLC34 proteins, which provides mechanistic insight into the transport cycle.