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Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergentinsoluble and assembled cytoskeleton. Indirect immunofluorescence and confocAl microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 1MI-abeled ankyrin can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of '2I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amioride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells.protein spectrin (4, 7-9), thus forming an integral membrane protein-ankyrin-spectrin-actin complex. To date, ankyrin and spectrin have been shown to associate with the anion exchanger (band 3) of erythrocytes, voltage-dependent Na' channels of neurons, lymphocyte adhesion antigen Pgp-1, ankyrin-binding glycoprotein 205 from brain, Na+/K+-

Experiments were performed to evaluate the effect of angiotensin II (ANG II) on the sodium transport activity of isolated intact rabbit proximal tubule cells. Initial rates of 22Na(+) uptake were measured in Na+-depleted and ouabain-treated cells in the presence of an opposing H+ gradient (pHin less than pHout). ANG II (10(-12)-10(-9) M) stimulated the initial rate of 22Na+ uptake by 33 +/- 2%, whereas amiloride (0.5 mM) inhibited both basal and ANG II-stimulated 22Na+ uptake. ANG II-stimulated rate of 22Na+ uptake was inhibited by the receptor antagonist saralasin. Additional experiments were performed to evaluate the effect of ANG II on the rate of recovery of pHin in acid-loaded proximal tubule cells. Cells were acid loaded by an NH4Cl pulse in the presence of the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. ANG II increased the initial rate of intracellular alkalinization, and this effect was inhibited by amiloride (1.0 mM). ANG II stimulation increased the Vmax of H+ efflux (from 0.53 +/- 0.02 to 0.64 +/- 0.04 pH units/min) without changing the Km for extracellular Na+. The present findings indicate that physiological concentrations of ANG II stimulate an amiloride-sensitive Na+-H+ antiport in proximal tubule cells.

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