Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix.

摘要

The proper targeting of ion channels to specialized domains is crucial for cell function. Kir4.1, the inwardly rectifying potassium channel, and aquaporin-4 (AQP4), the type 4 water-permeable channel, are localized at the basolateral domain of polarized epithelial cells; however, the mechanisms involved in their localization have yet to be determined. In this study, we investigated the role of the extracellular matrix in the localization of these channels in polarized Madin-Darby canine kidney (MDCK) cells. MDCK cells expressing green fluorescent protein-Kir4.1 or -AQP4 were cultured on laminin-1 or fibronectin and examined by confocal microscopy and cell surface biotinylation to assess plasma membrane expression of Kir4.1 and AQP4. Our data show that laminin-1 and fibronectin induce a significant increase in cell surface expression of both channels at the basolateral domain. Using fluorescence recovery after photobleaching, we demonstrate that laminin-1 and fibronectin reduce the diffusion rates of these channels. Finally, we show that the laminin receptor dystroglycan is important for cell surface expression of Kir4.1 but not AQP4. However, laminin-1 increases cell surface expression of both channels in cells deficient for dystroglycan, indicating that other receptors are involved. Indeed, RGD-containing peptides, which inhibit fibronectin binding to certain integrins, prevent the fibronectin-induced increase in Kir4.1 and AQP4 cell surface expression and reverse the laminin- and fibronectin-induced reduction in both channels' diffusion rates. Similarly, the alphavbeta3-integrin function-blocking antibody alters the reduction of AQP4 diffusion rates induced by both laminin and fibronectin, suggesting that alphavbeta3-integrin plays a role in the stabilization of APQ4 at the basolateral domain of epithelial cells.