Molecular determinants of pH sensitivity of the type IIa Na/P-i cotransporter

Type II Na/P-i cotransporters play key roles in epithelial P-i transport an d thereby contribute to overall P-i homeostasis, Renal proximal tubular bru sh border membrane expresses the IIa isoform, whereas the IIb isoform is pr eferentially expressed in small intestinal brush border membrane of mammals . IIa and Hb proteins are predicted to contain eight transmembrane domains with the N- and C-terminal tails facing the cytoplasm. They differ in their pH dependences: the activity of IIa increases at higher pH, whereas the Db shows no or a slightly opposite pH dependence. To determine the structural domains responsible for the difference in pH sensitivity, mouse IIa and II b chimeras were constructed, and their pH dependence was characterized, A r egion between the fourth and fifth transmembrane domains was required for c onferring pH sensitivity to the IIa-mediated Na/P-i cotransport. Sequence c omparison (IIa versus IIb) of the third extracellular loops revealed a stre tch of three charged amino acids in IIa (RER) replaced by uncharged residue s in IIb (GNT). Introduction of the uncharged GNT sequence (by REK) in IIa abolished its pH dependence, whereas introduction of the charged REK stretc h in Tm (by GNT) led to a pH dependence similar to IIa. These findings sugg est that charged residues within the third extracellular loop are involved in the pH sensitivity of IIa Na/P-i cotransporter.