Slc26A9 is a Sodium‐Activated Phosphate Transporter

Abstract

Inorganic phosphate homeostasis mainly relies on the activity of Pi transporters in the intestinal and renal epithelia. Most scientific efforts have been devoted to sodium-dependent transporters, while sodium-independent Pi transport systems have received much less attention. In order to identify new sodium-independent Pi transporters, members of the Slc4 and Slc26 families were expressed in Xenopus laevisoocytes. Among them, the Slc26A9 member induced a Pi uptake that was, once again, sodium-dependent or sodium-activated, given that in the absence of sodium no difference was observed in the water-injected oocytes, and sodium could not be replaced by potassium, lithium, or choline. Pi transport was, however, partially dependent on chloride, because when chloride was replaced by D-gluconate the uptake dropped by 40%. Dose-response and time-course assays of Slc26A9 expression did not show the classical relationship observed in type II and type III sodium-dependent Pi transporters, given that maximal transport (2-3 times above water-injected oocytes maximum) was reached with only 1 ng cRNA per oocyte and 2 days of expression. Pi uptake was also affected by pH. As in the case of type III Pi transporters, namely PiT1 and PiT2, higher Pi transport was observed at acid pH, and it decreased with alkalinity. Pi uptake was completely inhibited by Pi itself, and with less intensity by arsenate. 40-50% inhibition was obtained with phosphonoformate, sulfate, bicarbonate, the anion exchange inhibitors DIDS and SITS, and with phloretin, while the chloride channel inhibitor niflumic acid did not have any effect. Despite the low expression of Pi uptake above the water-injected level, an approximation to kinetic characterization was performed. A sodium activation kinetic revealed no saturation up to 100 mM, and it resulted in a provisional Hill coefficient of 1.3. For a Pi saturation curve, a net Km of 221 µM Pi was obtained. Expression in rat jejunum and kidney cortex was determined by real-time PCR and western blot (WB). To study the role of Slc26A9 in controlling Pi homeostasis, the protein abundance of this transporter was determined by WB in brush border membrane vesicles (BBMV) from the jejunum and kidney of rats that were fed with diets of differing Pi content. The animals were fed for five days, either ad libitum or during 4 hours in the morning, with fodders containing either 0.1 or 1.2% Pi. For some of the animals fed under the four-hour regime, their food was switched on the last day to study the change from either a 0.1% to a 1.2% Pi content or from a 1.2% to a 0.1% Pi content. WB results revealed that the abundance of Slc26A9 in jejunum or kidney cortex BBMV was not modified by the different Pi content in the diet, by the feeding regime, or by the acute dietary changes. In conclusion, Slc26A9 is a novel Pi transporter that seems to have limited relevance in the regulation of Pi homeostasis through its activity in the jejunum and the kidney. Nevertheless, the role of Slc26A9 in the lung, brain, stomach, and other tissues of high expression remains to be clarified.