Identification and characterization of PDZ proteins that differentially coordinate basolateral membrane expression of a CCD inwardly rectifying potassium channel, Kir 2.3

Abstract

The polarized expression of disparate transport proteins on two distinct membrane domains is an essential prerequisite for the vectorial transport of water, solutes and ions across epithelia. The renal cortical collecting duct, the site of potassium secretion in the kidney, provides a salient example. In these cells, the asymmetric expression of weakly inward rectifying potassium channels on the apical membrane and strongly rectifying potassium channel on the basolateral membrane increases the fidelity of the secretory process, ensuring that potassium preferentially exits the cell across the apical membrane into the lumen of the tubule in concert with the demands of potassium homeostasis (Giebisch, 1998). The identification of a plausible gene candidate, Kir 2.3, for the basolateral potassium channel (Welling, 1997) provided the impetus to elucidate the basis for polarized membrane targeting of a native channel (Le Maout et al., 1997). The basolateral membrane sorting determinant of Kir 2.3 is comprised of a unique arrangement of trafficking motifs, containing juxtaposing biosynthetic targeting and PDZ-based signals (Le Maout et al., 2001). In the present study, the mechanism by which the PDZ interactions coordinate the basolateral membrane expression of Kir 2.3 was elucidated. In contrast to apical missorting of Kir 2.3 channels lacking the basolateral sorting domain (Le Maout et al., 2001), deletion of the downstream PDZ binding domain causes channels to accumulate in an endosomal compartment. To identify PDZ proteins that functionally interact with the Kir 2.3 sorting signal, the yeast two-hybrid interaction system was employed. Two PDZ proteins that differentially regulate sorting of Kir 2.3 were identified. Consistent with a retention mechanism, one of these PDZ proteins, hLin-7b, couples Kir 2.3 to a multimeric scaffolding complex at the basolateral membrane in epithelial cells. The second PDZ protein, MOPP, has unique structural properties, allowing it to function as a natural dominant-negative PDZ protein. MOPP competes with hLin-7b for interaction with Kir 2.3, thereby regulating basolateral membrane expression of the channel. In conclusion, we propose that basolateral membrane expression of Kir 2.3 is coordinated by the sequential use of different sorting machinery in a multi-step basolateral sorting program.