FUNCTIONAL EXPRESSION OF RAT RENAL NA P-I-COTRANSPORT (NAPI-2) IN SF9CELLS BY THE BACULOVIRUS SYSTEM/

The recently cloned Na/P-i-cotransport system NaPi-2 is an apical memb rane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further fun ctional/structural studies, this transport system has been expressed i n Sf9 insect cells using a recombinant baculovirus, Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P-i-cotra nsport up to 20- to 50-fold over noninfected Sf9 cells. Transport of p hosphate in infected cells was highly dependent on sodium, exhibited a K-m for P-i of 0.114 mM and an apparent K-m for Na of 63 mM (Hill coe fficient of approximately 3) and was stimulated by high external pH. I nfected cells expressed a polypeptide of 65 kDa representing a nonglyc osylated form of the 85 kDa mature NaPi-2 transporter as present in pr oximal tubular brush-border membranes. By confocal microscopy expressi on of NaPi-2 protein was observed in the plasma membrane, yet submembr anous accumulation of NaPi-2 protein could not be excluded. This demon strates that the rat proximal tubular Na/P-i-cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics simil ar to that in isolated brush-border membranes. The 6His tagging will p ermit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.