Topology of extracellular loops of the type IIa Na/Pi-cotransporter and their contribution to the cotransport function

Colin Ehnes, Katja Kohler, Andrea Bacconi, Jürg Biber, Heini Murer and Ian C Forster.
Institute of Physiology, University of Zürich

The type IIa Na/Pi-cotransporter (NaPi-IIa) plays an essential physiological role in mammalian phosphate homeostasis by reabsorbing inorganic phosphate (Pi) in the kidney. It transports one divalent Pi molecule with 3 Na-ions per transport cycle, driven by the inward Na-gradient and negative membrane potential. Elucidating the structure-function relationships of NaPi-IIa is therefore of fundamental importance to understand its transport mechanism in vivo. To identify functionally important sites and their topological orientation, we use the substituted cysteine accessibility method (SCAM) to NaPi-IIa expressed in Xenopus oocytes. Novel cysteines are substituted at likely functionally important sites and the effect on function before and after exposure to cysteine modifying methanethiosulfonate (MTS) reagents is monitored by uptake or electrophysiological assays. Using SCAM, we previously established the importance of two opposed linkers between predicted transmembrane domains (Kohler et al., J. Gen. Physiol., 2002: 120:693-705) in contributing to a putative transport pathway through NaPi-IIa. Here we investigate if the first and last predicted extracellular linkers (ECL-1, ECL-4) are also functionally important, as they are well conserved among NaPi-II isoforms. Altogether 9 cysteine substitutions in ECL-1 and 6 in ECL-4 were made, most of which resulted in functional mutant cotransporters that were also surface biotinylatable by externally applied MTSEA-biotin. This finding, together with assays using impermeant MTS reagents, confirmed their extracellular orientation. Repeated exposure of mutants to fixed concentrations of MTS reagents established that the apparent accessibility of substituted cysteines varied over three decades, with those in ECL-4 being less reactive than those in ECL-1. Morever, at completion of the Cys-MTS reaction, only partial loss of transport function was documented, which was due to altered voltage dependency of the mutants. In particular, mutants G134C (ECL-1) and M533C (ECL-4) showed a reciprocal behavior in their voltage dependency that changed after MTS exposure: G134C had a weak voltage dependency that became WT-like after MTS exposure, whereas M533C showed the opposite behavior. Taken together, these findings suggest that the linkers are not part of the cotransport pathway, but, due to their different localization, they may interact with different parts of the voltage sensing elements that confer electrogenicity to NaPi-IIa.