Myosin VI mediates the movement of NHE3 down the microvillus in intestinal epithelial cells

Abstract

The intestinal brush border (BB) Na+/H+ exchanger, NHE3, is acutely regulated through changes in its endocytosis/exocytosis. Myosin VI, a minus-end directed motor, has been implicated in endocytosis at the inter-microvillar (MV) cleft and vesicle remodeling in the terminal web. We asked if myosin VI also regulates NHE3 movement down MV. Basal NHE3 activity and surface amount, determined by BCECF/fluorometry and biotinylation, respectively, were increased in myosin VI knock-down (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immuno-EM results showed NHE3 preferentially localized in the basal half of MV in control but in the distal half of myosin VI KD cells. Dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis; but NHE3's distribution along the MV was intermediate in dynasore-treated as compared to either myosin VI KD or untreated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells and suggest that myosin VI also moves NHE3 down MV.