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

Abstract

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 αvβ3-integrin function-blocking antibody alters the reduction of AQP4 diffusion rates induced by both laminin and fibronectin, suggesting that αvβ3-integrin plays a role in the stabilization of APQ4 at the basolateral domain of epithelial cells.

Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes

“…Our previous finding that exogenous laminin triggers a gain in the expression of AQP4 at the basolateral domain of epithelial MDCK cells when it is present in the culture substrate [25] prompted us to first investigate if laminin would exert a similar effect in astrocytes. Utilizing a biotinylation approach, we determined that primary astrocytes cultured in the presence of exogenous laminin, compared to untreated controls, did indeed exhibit an approximately threefold (2.97 ± 0.37) increase in AQP4 amounts at the plasma membrane (Fig 1A left and B).…”

“…Further, AQP4 clustering in astrocytes is significantly reduced in instances where DG expression has been disrupted via siRNA. We have additionally observed that laminin-induced clusters of AQP4 and its associated membrane domains exhibit significantly reduced lateral mobility, as measured using the fluorescence recovery after photobleaching assay [24] and a similar reduction of AQP4 mobility was observed in MDCK cells cultured on laminin as well [25]. Based on these data, it has been proposed that the interaction between the DGC and the extracellular matrix (ECM) plays a key role in tethering AQP4 in place at the perivascular astrocyte endfeet.…”

“…However, the highly asymmetric concentration of AQP4 in astrocyte endfeet may be mediated by mechanisms beyond this alone. Indeed, in the aforementioned study, we established via cell-surface biotinylation that the diffusional retardation caused by laminin in MDCK cells is accompanied by an increased expression of AQP4 at the basolateral plasma membrane [25], implying that laminin-DG interaction could regulate the expression levels of AQP4 at the plasma membrane by altering its internalization. A study published by Zhan et al .…”

Effects of aquaporin 4 and inward rectifier potassium channel 4.1 on medullospinal edema after methylprednisolone treatment to suppress acute spinal cord injury in rats

“…The water balance regulatory roles of AQP4 were also closely related to Kir4.1 21 . Nagelhus 22 found strict colocalization between AQP4 and Kir4.1, and Amiry 23 confirmed that AQP4-mediated water molecule transportation was related to the siphoning of K+.…”

Cell culture models and animal models for studying the patho-physiological role of renal aquaporins

“…In the same study, the authors also identified the signal involved in AQP4 lysosomal targeting/degradation. Recent in vitro studies in MDCK cells have shown how proteins in the ECM regulate the cell surface expression of AQP4 [131]. One of the most intriguing aspects of AQP4 organization within the plasma membrane is the presence of orthogonal arrays of particles (OAPs) in all sites where AQP4 is expressed, including principal cells in the kidney, airways epithelium, gastric parietal cells, astrocytes and skeletal muscle plasmalemma [151].…”