Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

Abstract

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).

Figure 1

Effect of intracellular ATP depletion on NHE activity and on total PIP₂ content. AP-1/NHE1′_HA cells were either untreated (Control) or metabolically depleted by incubation with 5 mM 2-deoxy-d-glucose and 5 μg/ml antimycin A for 10 min at 37°C. (A) Intracellular ATP content determined using luciferin–luciferase. Results were normalized to the control and are the mean ± SE of four determinations. (B) Measurement of intracellular pH (pH_i) using BCECF. The cells were acid-loaded by prepulsing with NH₄ ⁺ and recording was started upon addition of Na⁺ to the bathing medium. Representative of four similar pH_i determinations. (C) Measurement of cytosolic Ca²⁺ using Fura-2. Where indicated by the arrow, purinergic receptors were activated by addition of 1 mM extracellular ATP. Representative of four similar determinations. (D) Determination of PIP₂ content. Lipids were extracted from control or ATP-depleted cells, and the PIP₂ content was measured by TLC of ³²P-labeled PIP₂ (two leftmost bars) or by immunological detection of PIP₂ on dot-blots (two rightmost bars) as described in Materials and Methods. The insets show representative experiments, while the main panel summarizes the results of three chromatography and four dot-blot assays, normalized to the control (mean ± SEM).

Figure 2

Assessment of plasmalemmal PIP₂ content using PH_PLCδ-GFP. AP-1/NHE1′_HA cells were transiently transfected with PH_PLCδ-GFP and analyzed by confocal microscopy. The height of the cells was estimated, and optical sections, obtained through the middle of the cells, were acquired. (A) Untreated cells. (B) Cells incubated with 10 μM ionomycin for 5 min. (C) Cells preincubated overnight with 5 mM neomycin. (D) Cells that were ATP depleted as in Fig. 1. Images shown in A–D are representative of four independent experiments. (E) Ratio of the fluorescence intensity determined at the outermost edge of the image (plasma membrane or outermost layer of the cytosol) divided by the fluorescence of an equivalent area measured near the center of the cell (cytosol). Data are means ± SEM of at least 20 determinations of each kind, from four similar experiments. Where absent, error bars are smaller than the lines limiting the bars.

Figure 2

Assessment of plasmalemmal PIP₂ content using PH_PLCδ-GFP. AP-1/NHE1′_HA cells were transiently transfected with PH_PLCδ-GFP and analyzed by confocal microscopy. The height of the cells was estimated, and optical sections, obtained through the middle of the cells, were acquired. (A) Untreated cells. (B) Cells incubated with 10 μM ionomycin for 5 min. (C) Cells preincubated overnight with 5 mM neomycin. (D) Cells that were ATP depleted as in Fig. 1. Images shown in A–D are representative of four independent experiments. (E) Ratio of the fluorescence intensity determined at the outermost edge of the image (plasma membrane or outermost layer of the cytosol) divided by the fluorescence of an equivalent area measured near the center of the cell (cytosol). Data are means ± SEM of at least 20 determinations of each kind, from four similar experiments. Where absent, error bars are smaller than the lines limiting the bars.

Figure 4

Effects of ATP depletion and of ionomycin on the exchange activity of NHE1Δ582. AP-1 cells stably transfected with NHE1Δ582 were either untreated (Control), ATP depleted as in Fig. 1, or incubated with either 2 or 10 μM ionomycin for 5 min. Acid loading and fluorimetric determination of pH_i were as in Fig. 3. Note that ATP depletion and treatment with ionomycin were performed during the final stages of BCECF loading. (A) Representative pH_i determinations. (B) Rates of recovery measured at pH_i 6.4 are summarized as means ± SEM of at least four determinations.

Figure 6

Binding of PIP₂ to NHE1-derived fusion proteins. (Top) Primary sequence of the juxtamembrane region of NHE1 (residues 506–576) that contains two potential PIP₂-binding domains. The wild-type (WT) sequence is shown at the top, highlighting the cationic residues within the putative PIP₂-binding sites 1 and 2. The synthetic peptide, which is used for competition experiments (residues 550–564), is bracketed. In mutant 1 (M1), the cationic residues in site 1 were mutated to alanine (second line). In mutant 2 (M2), the cationic residues in site 2 were mutated to alanine (third line). In the double mutant (M1 + 2), both sets of cationic residues were mutated (bottom line). (bottom left) Graph showing results of PIP₂ binding determinations to either GST alone (leftmost bar) or GST fusions encompassing the indicated regions of NHE1. Where specified, sites 1 and/or 2 were mutated as above. Where indicated, peptide 550–564 was added to compete for PIP₂ binding. Results are normalized to the binding to GST, and are means ± SEM of eight determinations. (bottom right) Schematic representation of the predicted topology of NHE1, indicating the location of the putative PIP₂-binding sites 1 and 2.

Figure 3

Effect of neomycin on F-actin distribution and on NHE1 activity. AP-1/NHE1′_HA cells were incubated overnight without or with neomycin (5 mM). (A and B) Cells were stained with rhodamine-phalloidin to visualize F-actin. Representative micrographs of untreated (A) or neomycin-treated (B) cells. (C) Measurement of NHE activity. Control and neomycin-pretreated cells were loaded with BCECF, acidified by prepulsing with NH₄ ⁺, and the Na⁺-induced alkalinization was measured fluorimetrically. Representative of four similar pH_i determinations.

Figure 3

Effect of neomycin on F-actin distribution and on NHE1 activity. AP-1/NHE1′_HA cells were incubated overnight without or with neomycin (5 mM). (A and B) Cells were stained with rhodamine-phalloidin to visualize F-actin. Representative micrographs of untreated (A) or neomycin-treated (B) cells. (C) Measurement of NHE activity. Control and neomycin-pretreated cells were loaded with BCECF, acidified by prepulsing with NH₄ ⁺, and the Na⁺-induced alkalinization was measured fluorimetrically. Representative of four similar pH_i determinations.

Figure 5

Effects of PH_PLCδ-GFP and PM-5′-phosphatase-GFP on NHE activity. COS-1 cells were transiently transfected with either PH_PLCδ-GFP or PM-5′-phosphatase-GFP. After 24 h, transfected cells were identified by visualizing GFP fluorescence. A neutral density filter was interposed in the light path, and the cells were stained with BCECF while on the microscope stage. Acid loading and fluorimetric determination of pH_i were as in Fig. 3. (A) Fluorimetric pH_i determinations in control (open circles) and PH_PLCδ-GFP–transfected cells (solid circles). Na⁺ was added after 45 s. Values are means ± SEM of at least five determinations. (B) Rates of pH_i recovery measured during the first minute after the addition of Na⁺ in experiments like that in A are summarized as means ± SEM for control (n = 21), PH_PLCδ-GFP (n = 5), or PM-5′-phosphatase-GFP (n = 3)–transfected cells.

Figure 7

Comparative analysis of Na⁺-induced pH_i recovery of AP-1 cells transfected with wild-type and mutant forms of NHE1 under control and ATP-depleted conditions. (A) Analysis of surface expression of NHE1′_HA by immunoblotting. AP-1 cells were stably transfected with wild-type or mutant (M1, M2, and M1 + 2) NHE1′_HA. The cells were treated with or without chymotrypsin (100 U/ml, 5 min) and whole cell extracts were analyzed by electrophoresis and immunoblotting with anti-HA antibody. Note that the amounts of protein loaded are not identical: NHE1 = 4 μg; NHE1-M1 = 4 μg; NHE1-M2 = 10 μg; and NHE1-M1 + 2 = 7 μg. The position of the fully glycosylated (∼105 kD, Mature) and incompletely glycosylated (∼80 kD, Immature) forms of full-length NHE1, and of the main proteolytic fragment (∼75 kD) are shown. The blot is representative of three similar experiments. (B) Comparison of the Na⁺/H⁺ exchange activity determined from the rate of H⁺ extrusion as in Fig. 3 in AP-1 cells transfected with wild-type or mutant forms of NHE1′_HA. The rates of Na⁺-induced alkalinization were recorded, normalized for plasmalemmal NHE1 expression, and displayed as a function of the pH_i. Data are means ± SEM of at least five determinations. Where absent, error bars are smaller than the symbol. (C) AP-1 cells transfected with wild-type or mutant forms of NHE1′_HA were either untreated or subjected to ATP depletion, as in Fig. 1. The rates of Na⁺-induced alkalinization measured at pH_i 6.4 are illustrated. Data are means ± SEM of at least four determinations.

Figure 8

Comparative analysis of rates of ²²Na⁺ influx of AP-1 cells transfected with wild-type and mutant forms of NHE1 under control and ATP-depleted conditions. AP-1 cells transfected with wild-type (WT) or mutant forms (M1, M2, and M1 + 2) of NHE1′_HA were either untreated (Con) or subjected to ATP depletion (−ATP). (A–D) The cells were clamped at the indicated pH_i, and the rates of ²²Na⁺ uptake were measured, as detailed in Material and Methods. (A) WT; (B) M1; (C) M2; and (D) M1 + 2. To facilitate comparison of the effects of mutating the PIP₂-binding sites, the rates of ²²Na⁺ influx of wild-type and the mutant forms of NHE1 were normalized to their respective plasmalemmal protein levels, and then expressed relative to the maximal uptake rate of wild-type NHE1. In B–D, the dashed line indicates the wild-type NHE1 profile in control cells. Data are means ± SEM of three separate experiments, each performed in quadruplicate. Where absent, error bars are smaller than the symbol.