Rho GTPases dictate the mobility of the Na/H exchanger NHE3 in epithelia: role in apical retention and targeting

Abstract

Proximal tubular reabsorption of filtered sodium by the sodium/hydrogen exchanger isoform 3 (NHE3), located on the apical membrane, is fundamental to the maintenance of systemic volume and pH homeostasis. NHE3 is finely regulated by a variety of hormones and by changes in ionic composition and volume, likely requiring redistribution of the exchangers. We analyzed the subcellular distribution and dynamics of the exchangers by generating an epithelial line expressing NHE3 tagged with an exofacial epitope, which enabled us to monitor exchanger mobility and traffic in intact cells. Using determinations of fluorescence recovery after photobleaching in combination with dynamic measurements of subcellular distribution, we found that, in renal epithelial cells, NHE3 exists in four distinct subcompartments: a virtually immobile subpopulation that is retained on the apical membrane by interaction with the actin cytoskeleton in a manner that depends on the sustained activity of Rho GTPases; a mobile subpopulation on the apical membrane, which can be readily internalized; and two intracellular compartments that can be differentiated by their rate of exchange with the apical pool of NHE3. We provide evidence that detachment of the immobile fraction from its cytoskeletal anchorage leads to rapid internalization. These observations suggest that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and, consequently, the rate of transepithelial ion transport. Regulation of the interaction of NHE3 with the actin cytoskeleton can therefore provide a new mode of regulation of sodium and hydrogen transport.

Fig. 1.

Characterization of MDCK cells expressing . (A-C) Immunofluorescence staining. MDCK- cells were stained with anti-HA antibodies before (A) and after (B) permeabilization to localize superficial and total NHE3. For reference, the location of the nucleus was revealed by staining with DAPI (C). Representative x vs. y and x vs. z confocal optical sections are illustrated. (D) Na⁺/H⁺ exchange activity determinations. The rate of recovery of the cytosolic pH from an imposed acid load was measured in either wild-type MDCK cells (+) or MDCK- cells (♦) after addition of extracellular Na⁺. Data are the mean ± SE of eight experiments. (E) Immunoblot of a lysate of MDCK- using anti-HA antibodies. The blot in E is representative of three experiments.

Fig. 2.

SEM of the apical surface of MDCK cells expressing NHE3. MDCK- cells were either left untreated (A) or were treated with TxB for 2 h (B), as described in Materials and Methods. The cells were then immunolabeled with anti-HA antibodies, followed by secondary antibodies conjugated to 18-nm gold particles. Samples were visualized by SEM, and the gold particles were identified by backscattering. Arrows point to gold particles located on microvilli and arrowheads to gold in the intermicrovillar space. Note the absence of gold particles in B. Micrographs are representative of multiple fields from three separate experiments. (Scale bars, 1 μm.)

Fig. 3.

FRAP determinations. (A) Strategy used to measure FRAP of NHE3. The putative transmembrane topology of NHE3 is illustrated, showing the predicted exofacial location of the triple HA tag. The diagram also shows labeling of the tag with monoclonal anti-HA F(ab) fragment and Alexa Fluor 488-conjugated goat anti-mouse F(ab) fragment. (B) Recovery of fluorescence after photobleaching of apical labeled as described above (♦) and of apical GFP-tagged FcR (○). (C) Recovery after photobleaching of apical labeled as described above (♦) and of apical GPI-GFP (○). Data in B and C were binned over defined time intervals, and the bars are means ± SE of 10 determinations.

Fig. 4.

Effect of TxB on NHE3 distribution and mobility. (A-F) x vs. z digital reconstruction of serial optical slices of immunostained MDCK- cells, obtained by using confocal microscopy, surface (red) and total (green) NHE3. In A-C, the cells were fixed after the indicated treatment (see below) and exposed to anti-HA antibody followed by red-labeled secondary antibody. Next, the cells were permeabilized and exposed again to anti-HA and then green-labeled secondary antibodies. In D-F, surface-exposed NHE3 was tagged by addition of anti-HA to live cells at 22°C for 45 min, followed by exposure to red-labeled secondary at 22°C for an additional 45 min. After the indicated treatment, the cells were fixed, permeabilized, and exposed again to anti-HA and then green-labeled secondary antibodies to reveal the total NHE3 population. Shown are untreated cells (A and D), cells treated with TxB as described in Fig. 2 (B and E), and K⁺-depleted cells treated with TxB (C and F). (G) Recovery of fluorescence after photobleaching of apical labeled as described in Fig. 3. Otherwise untreated cells (♦) are compared with cells that had been K⁺-depleted and treated with TxB (○). Data were binned as in Fig. 3, and the bars are means ± SE of at least 12 determinations.

Fig. 5.

Proposed model depicting the subpopulations of NHE3 in epithelia. I, Immobile fraction of NHE3 at the apical membrane. The mobility of this subpopulation is restricted by anchorage to the actin cytoskeleton. II, Mobile fraction of NHE3 at the apical membrane. This subpopulation is likely to enter coated pits more readily than the immobile fraction. III, Rapidly recycling endomembrane fraction. IV, Endomembrane fraction of NHE3 that is not readily accessible from the external milieu. This fraction may exchange with pool III slowly, if at all.