Carlos' paper

Carlos' paper Regulation of Na,K-ATPase transport activity by protein kinase C

Carlos H. Pedemonte¹*, Thomas A. Pressley², Mustafa F. Lokhandwala¹ and Angel R. Cinelli³

¹College of Pharmacy, University of Houston, Houston, TX 77204-5515, ²Dept. of Physiology, Texas Tech University, Health Sciences Center, Lubbock, TX 79430, and ³Dept. of Anatomy and Cell Biology, SUNY at Brooklyn, NY 11203.

SUMMARY:

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase alpha-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the alpha-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase alpha-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the alpha-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent alpha-1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type alpha-1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the alpha-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the alpha-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC.

Key words: Na⁺,K⁺-ATPase; Na⁺-pump; Na⁺-transport

INTRODUCTION

The Na⁺,K⁺-ATPase transports Na⁺ and K⁺ ions across the plasma membrane of most eukaryotic cells and plays a key role in cellular ionic homeostasis (Glynn and Karlish, 1975; Kaplan, 1983; Jorgensen and Andersen, 1988). In renal epithelial cells, which are responsible for urinary Na⁺ reabsorption and extracellular fluid volume homeostasis (Simmons and Fuller, 1985; Garty and Benos, 1988), the Na⁺,K⁺-ATPase provides the driving force for vectorial Na⁺ transport from the lumen of the tubule to the blood supply (Soltoff and Mandel, 1984; Katz, 1988; Stanton and Kaissling, 1989). The minimum functional Na⁺,K⁺-ATPase molecule is a heterodimer composed of an alpha-subunit (Mr ~ 100 KD) and a heavily glycosylated beta-subunit (Mr ~ 50 KD) (Pedemonte and Kaplan, 1990).
In recent years, an increasing number of publications have suggested the short-term regulation of kidney Na⁺,K⁺-ATPase by hormones and intracellular second messengers (Bertorello et al., 1991; Aperia et al., 1992; Bertorello and Katz, 1993; Middleton et al., 1993; Aperia et al., 1994; Fisone et al., 1994). Several reports indicate that renal Na⁺,K⁺-ATPase activity may be regulated by phosphorylation/dephosphorylation processes (Bertorello and Katz, 1993). Both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) appear to phosphorylate the Na⁺,K⁺-ATPase (Bertorello et al., 1991; Middleton et al., 1993; Fisone et al., 1994; Lowndes et al., 1990; Chibalin et al., 1992). Depending on the tissue studied and the experimental conditions, stimulation of PKC leads to inhibition (Bertorello et al., 1991; Middleton et al., 1993; Vasilets et al., 1990; Satoh et al., 1993) or activation (Middleton et al., 1993;Schuster et al., 1984; Lynch et al., 1986; Hootman et al., 1987; Beach et al., 1987; Wang and Chan, 1990; Liu and Cogan, 1990) of Na⁺,K⁺-ATPase activity.
Working independently, both Beguin et al. (1994) and Feschenko and Sweadner (1995) have reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase alpha-1-subunit are phosphorylated by PKC. This result would suggest that the alpha-1-subunit NH₂-terminus is involved in regulation of the Na⁺,K⁺-ATPase activity. However, the fact that Feschenko and Sweadner (1994) did not observe any effect of phosphorylation on Na⁺,K⁺-ATPase activity raises the possibility that phosphorylation of the NH₂-terminus by PKC has no physiological significance. In this report, we show that stimulation of PKC by phorbol esters leads to activation of ouabain-sensitive 86Rb⁺-transport and reduced intracellular Na⁺ concentration ([Na⁺]i) of OK cells. The fact that these effects were not observed in cells expressing a NH₂-deletion mutant of the alpha-subunit support the hypothesis that the alpha-subunit NH₂-terminus plays a role in the regulation of Na⁺,K⁺-ATPase activity.

EXPERIMENTAL PROCEDURES:

Materials: Cell culture supplies were purchased from Gibco and Hyclone. Molecular biology reagents were from New England Biolabs, Dupont, Promega and USB. Ouabain was purchased from Calbiochem. Phorbol esters, phorbol 12-myristate 13-acetate (PMA) and 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) were obtained from Sigma Chemical Co. Other reagents were of the highest quality available.

Cell culture and transfection: The expression vector pCMV containing the rodent Na⁺,K⁺-ATPase alpha-1-subunit cDNA was obtained from Pharmingen. The preparation of the expression vector (myc/1.32) that encodes a shortened mutant of the alpha-1-subunit was described by Shanbaky and Pressley (1994). This vector expresses a rodent alpha-subunit in which the first thirty-one amino acids of the nascent polypeptide are replaced by an initiation methionine and a sequence of 10 amino acids (EQKLISEEDL) from the human c-myc oncogene product.
OK cells were maintained at 37ºC (10% CO2) in Dulbecco¹s modified Eagle¹s medium with 10% calf serum and antibiotics (DMEM-10). Plasmids containing the wild-type and mutant alpha-subunits were transfected into OK cells using liposomes. Cationic liposomes were prepared by sonication with 1 mg dioleoyl-L-alpha-phosphatidylethanol-amine and 0.4 mg dimethyl-dioctadecyl-ammonium bromide as indicated by Rose et al. (1991). The day before transfection, OK cells were seeded in the wells of a 96-well plate (3500 cells/well). The following day, the cells were transfected in 50 µl of Opti-MEM I containing 3 µg/ml of total DNA and 15 µl/ml of liposomes. The Na⁺,K⁺-ATPase of mock-transfected cells (vector alone, or vector plus liposomes, or liposomes alone) had the same activity and sensitivity to ouabain as non-treated host cells. Five hours after transfection, 200 µl/well of DMEM-10 was added. Two days later, cells were transferred to a medium containing 1 µM ouabain. Since the endogenous Na⁺,K⁺-ATPase of OK cells is sensitive to this level of ouabain, only OK cells that express the Na⁺,K⁺-ATPase containing the rodent alpha-subunit would be able to survive. After 10 days, cells from the wells that have single colonies were transferred to a medium containing 10 µM ouabain to select for those cells expressing the highest level of rodent alpha-subunit. Resistant colonies were expanded and maintained in DMEM-10 containing 10 µM ouabain.

Preparation of crude plasma membranes from OK cells: OK cells were harvested by mild trypsinization and suspended in lysis buffer (10 mM imidazole, 1 mM EDTA, pH 7.5). The cells were probe-sonicated twice for 15 sec with a 15 sec interval in an Ultrasonic homogenizer 4710 (Cole Parmer) at 25 watts and 80% power output. Samples were maintained in an ice-water bath during sonication. The suspension was centrifuged for 4 min. at 1,500 x g. The resulting supernatant was collected and centrifuged at 513,000 x g for 15 min. at 2 ºC (Beckman Optima TLX ultracentrifuge). The pellet was resuspended with a small volume of lysis buffer and used to determine protein and Na⁺,K⁺-ATPase activity. Protein was determined by the bicinchoninic acid method (Pierce Chemical) using BSA as standard.

Determination of Na⁺,K⁺-ATPase: Protein aliquots (2 mg/ml) were treated with 0.7 mg/ml SDS in the presence of 3 mM ATP and 10 mM imidazole, 0.4 mM EDTA, pH 7.5 for 10 min. at room temperature. Then, protein samples were put in an ice-water bath and BSA was added to a final concentration of 0.4 mg/ml. The SDS treatment was determined to be optimal for exposing latent Na⁺,K⁺-ATPase activity (Pedemonte, 1995). The Na⁺,K⁺-ATPase assay medium contained 0.05 mg/ml membrane protein; 0.3 mg/ml BSA and (mM): EGTA, 0.5; NaCl, 130; KCl, 20; MgCl2, 4; ATP, 3; imidazole, 50; pH 7.3. Enzymatic activity was determined as previously described (Pedemonte and Kaplan, 1986) at 37 ºC for 30 min. from the difference between the ATP hydrolysis measured in the absence and presence of ouabain. Concentrations of ouabain in the original dilutions were determined from the absorption at 221 nm by using a value of 15.5 x 103 for the molar extinction coefficient. Experiments were carried out in duplicate and repeated at least five times. Results are the average of at least five experiments ± SEM.

Determination of Rb⁺-transport: The experiments were performed with cells seeded at about 60% confluence in 24-well plates. To facilitate access of introduced ligands to the Na⁺,K⁺-ATPase, cells grown on plastic were exposed to culture medium containing EGTA prior to the measurement of Rb⁺-uptake (Contreras et al., 1989). No cell detachment from the plastic was observed during the 30 min. incubation. EGTA was not present during treatment with phorbol esters and Rb⁺-transport assay. However, we have determined that the stimulation of Rb⁺-transport by phorbol 12-myristate 13-acetate (PMA) was not affected by the presence of EGTA (data not shown).
To measure Rb⁺-transport, transfected cells (1 x 105 cells/well of a 24-well plate) were transferred to serum-free DMEM containing 50 mM Hepes (DMEM-Hepes), 2 mM EGTA and 10 µM or 10 mM ouabain (incubation medium). Cells were incubated for 20 min. at 37ºC in an air atmosphere and 10 min. at room temperature before addition of 1 µM phorbol ester. Five min. later, a trace amount of [86Rb⁺]-RbCl was added. Rb⁺-uptake was terminated after 20 min. by washing the cells four times with ice-cold saline. Cells were dissolved with SDS, and accumulated radioactivity was determined. Na⁺,K⁺-ATPase mediated Rb⁺-transport was estimated from the difference in tracer uptake between samples incubated in 10 µM and 10 mM ouabain. For non-transfected OK cells, Rb⁺-transport was measured in the absence and presence of 10 mM ouabain.
Since phorbol esters were dissolved in DMSO, the same amount of solvent was added to control samples. The amount of solvent used did not alter the Rb⁺-transport of control samples (data not shown). Each experiment was repeated at least four times

Determination of intracellular Na⁺ concentration: Fluorescence measurements of [Na⁺]i were performed using the membrane-permeant tetra(acetoxy-methyl) ester of the Na⁺-binding dye benzofuran-isophthalate-acetoxymethylester (SBFI-AM, Molecular Probe, Inc.) following standard protocols (Moore and Fay, 1993; Harootunian et al., 1989). Cells were loaded for 2 h. with the dye at room temperature in a serum-free HEPES medium (DMEM-50 mM Hepes; pH 7.4) containing 2-5 µM SBFI-AM and 0.1 % wt/vol Pluronic F-127 (Molecular Probe, Eugene,Ore). After loading, the cells were washed several times in DMEM-50 mM Hepes and incubated 30 min. in the same medium to allow de-esterification of SBFI-AM. The complete hydrolysis of SBFI-AM to SBFI was judged by changes in the excitation and emission spectra (Harootunian et al., 1989). Optical measurements were performed in serum-free HEPES buffered medium.
The optical setup consists of an upright epi-illumination microscope (Nikon Epiphot) with a video camera (MV-1070; Marshal Elect. CA) in the photographic port. [Na⁺]i was calculated according to the equation described by Grynkiewicz et al. (1981) with a Kd of SBFI for Na⁺ of 18 mM (1989). Other terms of the equation were assessed by in situ calibration. No significant level of autofluorescence was observed in the cells, and the concentration of reagents added to the cell medium did not affect the fluorescence levels as judged by determinations performed at an excitation wavelength corresponding to the isosbestic point of SBFI (370 ± 2.5 nm; Omega Optical, Brattleboro, VT). In the pictures, [Na⁺]i changes are illustrated by pseudocolors resulting from subtracting the basal level of [Na⁺]i from those obtained after addition of PMA. The basal [Na⁺]i was the same in cells transfected with the wild type and mutant alpha-1 cDNA.
At the end of each experiment, in situ calibration of the excitation ratio of SBFI was performed to accurately assess [Na⁺]i. After permeabilization with gramicidin D (10 µM), cells were superfused with different Na⁺ concentrations. Calibration curves of [Na⁺]i were the same for cells transfected with both plasmids.

Statistics: Comparisons between groups were performed by Student¹s t-test for unpaired data.

RESULTS

Expression of the NH₂-deletion mutant Na⁺,K⁺-ATPase in OK cells: Many of the studies examining the regulation of the Na⁺,K⁺-ATPase by hormones and second messengers have been performed in isolated rat proximal tubule segments or dissociated rat proximal tubule cells (Bertorello and Katz, 1993). The OK cells used in our experiments are an established epithelial cell line which is often studied as a physiological model system of renal proximal tubule function (Malstrom et al., 1987; Nash et al., 1993). Rb⁺ was used as a K⁺ congener to determine the transport activity of the Na⁺,K⁺-ATPase. We found that both the ouabain-sensitive and ouabain-insensitive Rb⁺-uptake were linear with respect to time for at least 30 min. (Fig. 1). Although this experiment was done with wild-type OK cells, similar results were obtained with the transfected cells described below.

Fig. 1: Incorporation of Rb⁺ into OK cells incubated at room temperature in the conditions described in ³Experimental². Rb⁺-transport was measured in the presence and absence of 10 mM ouabain. Ouabain-sensitive (triangles) and ouabain-insensitive (squares) transports are represented. Data presented is the average of two experiments carried out in triplicate.

To investigate the functional role of the alpha-subunit NH₂-terminus in regulation of enzymatic activity by PKC, we transfected OK cells with wild-type rodent kidney alpha-1 cDNA or a mutant cDNA that encodes a shortened form of the alpha-subunit. Expression of rodent alpha-1 cDNA would be expected to confer resistance to 10 µM ouabain, which would be otherwise lethal for non-transfected cells. As previously shown in COS-1 cells (Shanbaky and Pressley, 1994), OK cells expressing the wild-type rodent alpha-1 or the NH₂-deletion mutant were resistant to 10 µM ouabain (Fig. 2). There was no difference in the growth rate between cells transfected with wild type alpha-1 cDNA and the NH₂-deletion mutant alpha-1 cDNA.

Fig. 2: Dependence of ouabain-sensitive Rb⁺-transport on ouabain concentration. Data are presented as percentage of remaining activity with respect to non-inhibited Na⁺,K⁺-ATPase. The Rb⁺-transport of wild-type host OK cells (squares), cells transfected with wild-type rodent alpha-1 cDNA (circles) and cells transfected with the NH₂-deletion mutant alpha-1 cDNA ( ) was determined as indicated in ³Experimental². Plotted are means ± s.e. (n = 4).

OK cells express an endogenous alpha-subunit with a high affinity for ouabain (Fig. 2). In contrast, cells expressing wild-type or mutant rodent alpha-1 were more resistant to the glycoside, consistent with the expression of a ouabain-resistant exogenous form (Price and Lingrel, 1988). There was no significant difference in the ouabain sensitivity between cells transfected with the alpha-1 cDNA and the NH₂-deletion mutant alpha-1 cDNA.
The same level of Na⁺,K⁺-ATPase activity was determined in transfected and non-transfected cell membranes (Fig. 3).

Fig. 3: Na⁺,K⁺-ATPase activity in non-transfected OK cells (OK-wt) and in cells transfected with the wild-type alpha-1 cDNA (alpha-1-wt) and the NH₂-deletion mutant alpha-1 cDNA (alpha-1-mut). Enzymatic activity was determined at saturating concentrations of all the enzyme ligands as indicated in ³Experimental².

Similarities in alpha-subunit abundance have been demonstrated by Shanbaky and Pressley (1994) in host and transfected COS-1 cells. These observations suggest that Na⁺,K⁺-ATPases containing the endogenous alpha-subunits have been replaced by Na⁺,K⁺-ATPases containing the ouabain-resistant wild-type or mutant rodent alpha-1.

Effect of PKC activation on Rb⁺-transport: We next evaluated the regulation of the endogenous and exogenous Na⁺,K⁺-ATPase by PKC. Cells were transferred to serum-free DMEM-Hepes medium containing different amounts of ouabain, [86Rb⁺]-RbCl, and 1 µM phorbol 12-myristate 13-acetate (PMA). Note that changes in the medium were held to a minimum to minimize any alterations in cellular homeostasis. The ouabain-sensitive Rb⁺-transport in non-transfected host OK cells was 11.6 ± 0.7 nmol/mg/min, which is almost equal to that measured by Middleton et al. (1993) in the same cells (49.8 ± 4.7 nmol/mg/4 min). Treatment of host cells with PMA increased Rb⁺-transport by 24 ± 3 % (Fig. 4).

Fig. 4: Effect of phorbol ester treatment on Rb⁺-transport mediated by the Na⁺,K⁺-ATPase of non-transfected OK cells (OK-wt), and cells transfected with the wild-type rodent alpha-1 cDNA (alpha-1-wt) and the NH₂-deletion mutant alpha-1 cDNA (alpha-1-mut). Data are presented as percentage of activity with respect to non-treated control. Treatments were performed as indicated in ³Experimental² and in the text. *P < 0.001.

Cells transfected with the rodent alpha-1 cDNA displayed a Rb⁺-transport of 9.5 ± 1.4 nmol/mg/min. This activity was measured in cells that were maintained in the presence of 10 µM ouabain during growth and assay. Under these conditions, endogenous Na⁺,K⁺-ATPase activity should have been negligible, and any ouabain-sensitive Rb⁺-transport measured should have been attributable to Na⁺,K⁺-ATPase containing the introduced rodent alpha-subunit. The Rb⁺-uptake of cells transfected with the rodent alpha-1 cDNA (alpha-1-wt) was increased 58 ± 5 % by treatment with PMA (Fig 4). On the contrary, 1 µM 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a phorbol ester that does not stimulate PKC, did not change the level of ouabain-sensitive Rb⁺-transport. This suggests that the activation induced by PMA was specific and mediated by PKC.
Beguin et al. (1994) and Feschenko and Sweadner (1995) determined that amino acids close to the NH₂-terminus of the Na⁺,K⁺-ATPase alpha-1-subunit are targets for PKC phosphorylation. It follows that the activation of Rb⁺-transport we observed may have been produced by phosphorylation within this region. To test whether the alpha-subunit NH₂-terminus plays any role in the modulation of Rb⁺-transport, the experiments described above were repeated using cells transfected with the NH₂-deletion mutant alpha-1 cDNA (alpha-1-mut). These cells displayed a Rb⁺-transport of 11.2 ± 1.9 nmol/mg/min, comparable to the level of transport measured in both non-transfected and wild-type rodent alpha-1-expressing cells. Thus, cells expressing the alpha-subunit NH₂-terminus deletion mutant have the same level of maximal Na⁺,K⁺-ATPase, ouabain-sensitive Rb⁺-transport, and similar concentration dependency to ouabain as cells transfected with the wild-type rodent alpha-1 cDNA. However, in sharp contrast to the endogenous and introduced wild-type enzymes, the activity of the mutant was not significantly modified by treatment with PMA (Fig. 4). The same result was observed with 4alpha-PDD, the phorbol ester that does not stimulate PKC (Fig. 4). Thus, an intact alpha-subunit NH₂-terminus was essential for activation of the Na⁺,K⁺-ATPase by PKC.

PMA treatment reduces the [Na⁺]i: An increased Na⁺,K⁺-ATPase activity should result in reduced [Na⁺]i. To test this possibility, the [Na⁺]i was determined in situ by changes in fluorescence produced by the Na⁺ indicator SBFI (Minta and Tsien, 1989). Transfected cells were loaded with the membrane permeant derivative of SBFI (SBFI/AM) and the level of emitted fluorescence upon excitation at 340 and 385 nm was monitored using a video imaging system, as previously described (Cinelli et al., 1995). PMA treatment of wild-type rodent alpha-1 transfected cells produced a reduction in [Na⁺]i (Fig. 5).

Fig. 5: Top: PMalpha-dependent reduction of [Na⁺]i revealed by fluorescence microscopy from cells transfected with the wild-type alpha-1 cDNA. Images correspond to 0 and 2 sec after the addition of 10 µM PMA. Pseudocolor calibration: dark pink, 0-5 mM; dark blue, 5-10 mM; light blue, 10-15 mM; green, 15-20 mM; yellow, 20-25 mM; red, 25-30 mM; white, above 30 mM. Bottom: Images from cells transfected with the NH₂-deletion mutant of the alpha-1 cDNA before and 2 sec after the addition of 10 µM PMA. The same result was observed after 60 sec.

Images of SBFI/AM-loaded cells (displayed in pseudocolor) obtained before and 2 seconds after addition of PMA illustrates the rapid change in [Na⁺]i. In situ calibration of the excitation ratio of SBFI at various [Na⁺]i indicated that PMA treatment reduced [Na⁺]i from 19.7 ± 2.4 (n = 42) to 5.6 ± 0.6 (n = 18) mM. Washing out the PMA from the cell medium restored the initial steady-state level of [Na⁺]i, and phorbol esters that do not stimulate PKC had no effect on the [Na⁺]i.
As previously observed with Rb⁺-uptake, the [Na⁺]i of cells expressing the NH₂-deletion mutant alpha-1 (17.2 ± 2.5 mM) was not affected by PMA (Fig. 5, bottom). This lack of response was not due to the cells being dead or damaged since a 15 mM reduction of [Na⁺]i was observed when the cells were treated with 1 mM 8-Br-cAMP, which stimulates protein kinase A, or 8 µM 5-(N-methyl-N-isobutyl)-amiloride, which inhibits the Na⁺/H⁺-exchanger. These reagents had the same effect on cells transfected with the wild-type alpha-1 cDNA. An increase in [Na⁺]i was observed when K⁺ was removed from the cell medium and following the application of ouabain (100 µM) in cells transfected with both plasmids (data not shown).

DISCUSSION:

In this study, we have demonstrated that the Na⁺,K⁺-ATPase mediated Rb⁺-transport is stimulated by activation of PKC and that the presence of the alpha-1-subunit NH₂-terminus is essential to observe the modulation of activity. It is important to notice that the lack of response to PMA observed in cells transfected with the alpha-subunit NH₂-deletion mutant was not due to a lowered ouabain-sensitive Rb⁺-transport activity. These cells have the same level of Rb⁺-transport, measured under in vivo conditions (intact cells), and maximal Na⁺,K⁺-ATPase, measured at saturating concentration of the ligands, as cells transfected with the wild-type rodent alpha-1 cDNA. This indicates that the total number of active Na⁺,K⁺-ATPase molecules is the same in both cell lines. In cells transfected with either the wild-type or mutant alpha-subunit cDNAs, the Na⁺,K⁺-ATPase activity was about 2.5 µmoles Pi/mg/h. This corresponds to 42 nmoles Pi/mg/min. In cells transfected with either the wild-type or mutant alpha-subunit cDNAs, the Rb⁺-transport was about 10 nmoles/mg/min. Since the Na⁺,K⁺-ATPase transports 2 Rb⁺ ions per ATP that is hydrolyzed, this level of Rb⁺-transport corresponds to an ATPase activity of 5 nmoles Pi/mg/min. Thus, the maximal ATPase activity was about 8 times higher than the ATPase activity associated with the Rb⁺-transport. Therefore, the Na⁺,K⁺-ATPase of intact cells transfected with the NH₂-deletion mutant cDNA was working at about 8 times lower velocity than Vmax and the lack of effect of phorbol ester treatment was not due to the pump working at Vmax.
Even though it has been shown that elimination of the alpha-subunit NH₂-terminus may produce a change in the equilibrium E1/E2 of the protein conformations (Jorgensen and Karlish, 1980) or affect the interaction between the cations and the enzyme (Wierzbicki and Blostein, 1993), these experiments were performed with purified protein or with enzyme ligand concentrations that were very far from an in vivo condition. It is therefore unclear if these findings can be extrapolated to the enzyme when operating in an intact cell. Our experiments, which were performed in an in vivo condition, suggest that the basic functioning of the Na⁺,K⁺-ATPase activity appears not to be altered by the elimination of the alpha-subunit NH₂-terminus. Therefore, this region may have no effect on the basal Na⁺,K⁺-ATPase activity but on the regulation of this activity. This paper is the first report showing in an in vivo condition that the NH₂-terminal end of the alpha-subunit is involved in the regulation of the Na⁺,K⁺-ATPase activity by PKC.
The same hormones that regulate Na⁺,K⁺-ATPase in proximal tubules (Bertorello and Katz, 1993; Aperia et al., 1994) appear to regulate the activity of the Na⁺/H⁺-exchanger which mediates Na⁺ entry into epithelial cells (Mahninsmith and Aronson, 1985; Grinstein and Rothstein, 1986; Nord et al., 1987; Gesek and Schoolwerth, 1991). Therefore, it can be argued that the observed increase of Rb⁺-uptake might be due not to regulatory modulation of the Na⁺,K⁺-ATPase, but to an increased [Na⁺]i produced by PKC mediated stimulation of the Na⁺/H⁺-exchanger. However, if this were true, Rb⁺-transport would have been similarly affected in cells expressing both the wild-type and the NH₂-deletion mutant. Furthermore, we have determined that upon treatment with PMA the [Na⁺]i was reduced in cells transfected with the wild-type rodent alpha-1 cDNA and not affected in cells expressing the NH₂-terminal deletion mutant alpha-1. Taken together, these results indicate that PKC has specifically stimulated the Na⁺,K⁺-ATPase activity, and there is no evidence suggesting that PKC has affected other proteins mediating Na⁺ transport. Our results are consistent with the observations that hormones with receptors coupled to stimulation of PKC increase Na⁺ reabsorption in proximal convoluted tubules (Schuster et al., 1984; Beach et al., 1987; Wang and Chan, 1990; Liu and Cogan, 1990). Stimulation of Na⁺,K⁺-ATPase activity with phorbol esters has been observed in rat proximal tubule cells (Ferraile et al., 1995), rat hepatocytes (Lynch et al., 1986), human lymphocytes (Norby and Obel, 1993), pancreatic acinar cells (Hootman et al., 1987) and peripheral nerve from diabetic rabbits (Lattimer et al., 1989).
Previous reports have identified Ser-11 and Ser-18 of the NH₂-terminal end of the Na⁺,K⁺-ATPase alpha-1-subunit as targets for phosphorylation by PKC (Beguin et al., 1994; Feschenko and Sweadner, 1995). Since Feschenko and Sweadner (1994) did not observe any effect of phosphorylation on Na⁺,K⁺-ATPase activity(1)(footnote), the relevance of the alpha-subunit NH₂-terminus in the regulation of Na⁺,K⁺-ATPase by PKC was an open question. Even though our results have not demonstrated explicitly phosphorylation of the alpha-subunit, such covalent modification seems a likely explanation for the increased Rb⁺-transport that we observed. However, we cannot rule out the possibility that PKC may phosphorylate other amino acids of the Na⁺, K⁺-ATPase subunits or even another protein. Nevertheless, independent of which amino acids or protein is phosphorylated, our results clearly indicate that amino acids of the alpha-subunit NH₂-terminus are involved in the PKC modulation of the Na⁺,K⁺-ATPase activity.
Recent reports of biochemical and electrophysiological studies have suggested that the NH₂-terminal segment may affect the interaction between cations and the enzyme (Wierzbicki and Blostein, 1993; Vasilets et al., 1991; Vasilets et al., 1993; Horisberger et al., 1993; Daly et al., 1994). Because it is a region of high sequence diversity among the alpha-subunits from various species and between the alpha-isoforms, the NH₂-terminus of the alpha-subunit has generated interest and speculation with respect to its role in Na⁺,K⁺-ATPase function. The most prominent feature of the NH₂-terminal segment is a high proportion of charged amino acids. The rodent alpha-1 has 12 positively charged, 8 negatively charged and 3 polar amino acids in the first 32 residues of the mature subunit. Between Ser-11 and Lys-32, all amino acids but two are charged or polar. The amino acid sequence, as well as Raman spectroscopy, suggests that the secondary structure of the NH₂-terminal segment corresponds to an alpha-helix (Lupas et al., 1991; Ovchinnikov et al., 1988).

Fig. 6: Representation of the rodent alpha-1 NH₂-terminal segment as an alpha-helical wheel. Polar (triangles), aliphatic (circles ), positively-charged (+) and negatively-charged (-) amino acids are included.

Fig. 6 illustrates amino acids 8-32 in an alpha-helical representation. A 26-residue region beginning just carboxy to Pro-7 is depicted. Pro-7 was excluded because it is not stable in the helical conformation and the maximal propensity to form alpha-helix was determined for amino acids 8 to 32 (Lupas et al., 1991). By examination of this putative alpha-helix, it is possible to distinguish some organization of the charged amino acids with respect to the serines that are the putative target of PKC (Fig. 6). There is a 90º face of the helix that contains only positive charges (sector A), and it is just opposite to the face containing Ser-11 and Ser-18 (sector B). The other two sectors of the helix contain an almost equal number of interdispersed positive and negative charges. This asymmetric distribution of charges may have functional relevance. This is represented schematically in Fig. 7, which may help to visualize a hypothesis that considers the distribution of charges and interprets some of the results previously obtained. This model assumes that serines 11 and 18 are located just in front of a group of negative charges in another arm of the alpha-subunit. Upon the addition of the bulky, negatively-charged phosphates by PKC, the NH₂-terminal segment may move by charge repulsion. Due to this movement, the positively charged face of the NH₂-terminal segment would then interact with other parts of the Na⁺,K⁺-ATPase molecule and lead to the modulation of enzymatic activity.

Fig. 7: Hypothetical scheme depicting the role of phosphorylation by PKC and the accumulation of positive charges on one face of the alpha-subunit NH₂-terminus.

In conclusion, we have shown that stimulation of PKC activates Na⁺,K⁺-ATPase in a kidney cell line. This activation involves amino acid(s) located in the NH₂-terminus of the alpha-subunit. Even though the presence of the alpha-subunit NH₂-terminus is not required for expression of the basic Na⁺,K⁺-ATPase activity , the differential interaction of the NH₂-terminus with other intracellular domains of the alpha-subunit may be a mechanism by which hormones regulate Na⁺,K⁺-ATPase activity.

Footnotes: (1) The lack of effect may be due to the authors determination of Vmax. It is likely that PKC may affect the affinity for Na⁺ rather than the Vmax (Feraille et al., 1995).

Acknowledgments: The authors want to thank Hemangini Joshi for expert technical assistance, Dr. John P. Middleton (Duke Univ.) for his advice on the measurement of Rb⁺-transport in OK cells and comments about the manuscript, and Drs. Douglas Eikenburg (Univ. of Houston) and Julius C. Allen (Baylor College of Medicine, Houston) for their critical reading of the manuscript. This work was supported by grants from the National Science Foundation (CHP), National Institute of Health (DK52273 to CHP, RR-19799 to TAP, and DC01804 to ARC) and a limited grant-in-aid from the University of Houston (CHP).

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Abbreviations: Na⁺,K⁺-ATPase and Rb⁺-transport refers to the same protein activity; PKC, protein kinase C; OK, opossum kidney; PMA, phorbol 12-myristate 13-acetate; 4 alpha-PDD, 4 alpha-phorbol 12,13-didecanoate.