From the * Department of Internal Medicine, and Department of Cellular and Molecular Physiology, Yale University School of
Medicine, New Haven, Connecticut 06510
By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase)
influences many aspects of cellular physiology. Of the four isoforms of the pump,
1 is ubiquitous,
2 is predominant in skeletal muscle, and
3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding
benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa
cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time
course of the decrease in [Na+]i after removing external Na+. All transfected rat
subunits were highly ouabain
resistant: the
1 isoform is naturally resistant, whereas the
2 and
3 isoforms had been mutagenized to render
them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 µM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human
1 isoform was 12 mM, which was similar to the Km of rat
1. The
2 and
3
isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing
3 had a high resting [Na+]i. The
maximal activity of native
1 in the
3-transfected cells was only ~56% of native
1 activity in untransfected HeLa
cells, suggesting that transfection with
3 led to a compensatory decrease in endogenous
1 pumps. We conclude
that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence
1 <
2 <
3. The
3 isoform
may be suited for handling large Na+ loads in electrically active cells.
The Na-K pump (Na,K-ATPase) has two main protein
subunits, and
. Four isoforms of the
subunit are
known, and three of the
. Of the
isoforms,
1 is
ubiquitous. It appears to be a "housekeeping" isoform
in most tissues, but is expressed at high levels in epithelia with high rates of solute transport. The other isoforms have a more restricted distribution:
2 is found
in muscle, adipose tissue, and brain;
3 is expressed in
neural tissue and heart; and
4 is present in testis. The
subunit is essential for normal targeting and correct
insertion of the
subunit into the cell membrane. In
addition, the
subunit appears to have some influence
on the catalytic properties of the enzyme, even though
it is the
subunit that has the binding sites for Na+, K+,
ATP, and ouabain ( Jaisser et al., 1994
).
Although little is known about 4, the other
isoforms
exhibit marked anatomic, developmental, and pathophysiologic diversity, suggesting that the
isoforms have
different functional roles. Experiments in which single,
defined
isoforms are transfected into cultured cells
show that rat
isoforms differ in their sensitivity to ouabain: rat
1 is ouabain resistant (inhibited only by millimolar ouabain concentrations), whereas rat
2 and
3
are ouabain sensitive (completely inhibited by 1-5 µM
ouabain). Nevertheless, work on transfected cells has
been limited by techniques available for assessing functional differences (e.g., differences in affinity for Na+)
among
isoforms. First, it is difficult to distinguish unambiguously the
2 and
3 isoforms in experiments on
mammalian cells, which invariably express
1. Second,
some assay systems consist of broken membranes, and
thus are not "sided." Third, even in sided preparations, previous transfection studies have not controlled intra-
and extracellular ion concentrations independently.
Such control is important because the Na-K pump rate
depends on intracellular sodium concentration ([Na+]i)
(Garay et al., 1973).
The objective of the present study was to measure the
apparent Km for [Na+]i of the rat 1,
2, and
3 isoforms, transfected into human HeLa cells. One approach for assessing pump function would be to measure Na,K-ATPase activity. Such assays, however, are usually performed in broken (i.e., "unsided") membranes.
Other approaches would be to measure isotopic fluxes
or pump current. However, [Na+]i usually cannot be
measured in such experiments; [Na+]i must be clamped
using a patch pipette or ionophores. Our approach was
to use the Na+-sensitive dye SBFI (sodium-binding benzofuran isophthalate)1 (Minta et al., 1989; Harootunian
et al., 1989
) to monitor [Na+]i, and from these data
compute a measure of the Na+ efflux (i.e., d[Na+]i/dt).
We exploit a technique proposed by Negulescu and
Machen (1990)
for Na+ loading cells and using SBFI to
monitor the subsequent rate of [Na+]i recovery (i.e.,
decrease).
We studied untransfected HeLa cells, as well as HeLa
lines stably transfected with various rat isoforms. All
cells expressed the ouabain-sensitive, native human
1
subunit, as well as the native human
1 subunit of the
Na-K pump. One line was stably transfected with rat
1,
which is naturally ouabain resistant. Two other lines
were transfected with rat
2 and
3 that had been mutagenized to render them ouabain resistant ( Jewell and
Lingrel, 1991
). Thus, we could use differential ouabain
sensitivity to distinguish the transfected, ouabain-resistant rat
isoforms from the native, ouabain-sensitive human
1 pumps. Others have approached the question
of whether the Na+ affinities of the pump isoforms are
different using these cell lines ( Jewell and Lingrel, 1991
;
Munzer et al., 1994
). We found that rat and human
1
isoforms have similar apparent Km values for [Na+]i,
but that rat
2 and
3 have progressively higher Km values. Moreover, the cells expressing
3 had a very high
resting [Na+]i.
Cells Studied
We used four HeLa-based cell lines ("Jewell-Lingrel cell lines"),
three of which were transfected with rat isoforms ( Jewell and
Lingrel, 1991
). All were kind gifts of Dr. Jerry Lingrel. The first
was untransfected HeLa cells, which express only the human
1
Na,K-ATPase isoform. This isoform is ouabain-sensitive (>95%
inhibition at 1 µM ouabain). The second HeLa cell line (
1-8G) was stably transfected with the unmodified, but naturally ouabain resistant, rat
1. The third line of HeLa cells (
2-2G) was transfected with a mutagenized rat
2 isoform of the Na-K pump, designated
2*. In this mutant isoform, two amino acid residues
were changed so as to confer ouabain resistance (>95% inhibition at 4 mM ouabain). The fourth HeLa cell line (
3-5G) was
stably transfected with a similarly mutagenized ouabain-resistant
rat
3, termed
3*.
Cells were grown in DMEM plus 10% FCS supplemented with
penicillin, streptomycin, and fungizone. The medium for the
three cell lines transfected with rat isoforms also contained 1 µM ouabain to inhibit the endogenous human
1 isoform, thereby
applying selection pressure.
Solutions
Table I summarizes the composition of our solutions, all of which were made with ultrapure water (PICOpure water systems; Hydro Research, Triangle Park, NC). The pH was adjusted with HCl or NMDG (N -methyl-D-glutamate). The solutions for calibrating the intracellular dye had [Na+] values between 0 and 160 mM, and were made by mixing calibration buffers 1 (solution 4) and 2 (solution 5). The acetoxymethyl ester of SBFI was obtained from Molecular Probes, Inc. (Eugene, OR), and DMSO (dimethyl sulfoxide) from J.T. Baker, Inc. (Phillipsburg, NJ). Other chemicals were supplied by Sigma Chemical Co. (St. Louis, MO).
Table I. Composition of Working Solutions |
Spectrofluorometry
We used a dual-beam spectrofluorometer (1681; SPEX Inc., Edison, NJ) to measure [Na+]i in a population of cells. Cells were grown on a 8-mm2 glass coverslip, which was inserted into a custom-designed ~0.8-ml quartz flow-through cuvette. A thermistor in the cuvette permitted continuous temperature monitoring. The cuvette was continuously superfused with prewarmed medium delivered at 2.2 ml/min through water-jacketed Tygon tubing.
We grew the cells to 80-100% confluence, and then serum-starved them (0.5% FCS) 12 h before study. On the morning of
the study, we loaded the cells with the Na-sensitive dye SBFI (Harootunian et al., 1989; Negulescu and Machen, 1990
) as follows.
The dye-loading solution was made by adding 7 µl DMSO and 7 µl of a Pluronic F-127 solution (20% in DMSO) to a vial containing 50 µg SBFI-AM. The vial was vortexed, and the contents diluted into 3 ml standard buffer (solution 1) at room temperature
(RT). Coverslips were incubated in this solution at RT for 3-4 h,
shielded from the light, with gentle agitation. Coverslips were
then washed in standard buffer for 30 min at RT with agitation. A
coverslip of dye-loaded cells was then inserted into the cuvette
and an excitation scan obtained (excitation from 320 to 400 nM,
emission at 505 nm). The coverslip was rejected if the excitation
spectrum was not a smooth, skewed, bell-shaped curve with a
peak intensity at ~355 nM (characteristic of SBFI-free acid) (Harootunian et al., 1989
), or if the peak intensity was not at least sixfold greater than that obtained from a coverslip of cells not
loaded with dye.
We calculated [Na+]i as follows. First, we obtained fluorescence intensities at 505 nm, while exciting at either 340 or 380 nm. These I340 and I380 values were acquired continuously and sampled every 1.5-4.0 s. Second, we corrected these I340 and I380 values for the autofluorescence at the corresponding wavelengths, and computed the corrected I340/I380 ratio. Third, we converted these I340/I380 ratios to [Na+]i values using the data obtained from a two-point calibration performed at the conclusion of each experiment. In this calibration, we determined the I340/I380 ratio while exposing the cells to calibration buffers containing, first, 90 mM Na+ and, then, 0 mM Na+. For both buffers, the sum [Na+] + [K+] was 160 mM. To equalize [Na+]i and [Na+]o, we also included in the calibration buffers the monovalent cation ionophores gramicidin D (10 µM), nigericin (10 µM), and monensin (14.5 µM).2 (We show below that this ionophore combination did in fact equalize [Na+]i and [Na+]o.) As described in RESULTS, we used the I340/I380 ratios thus obtained at [Na+]i = 0 and 90 mM to convert the I340/I380 ratios obtained earlier in the experiment to [Na+]i values.
Although the majority of our experiments were done with the
standard two-point calibration protocol described above, in the early stages of the project we performed some experiments with calibration procedures that differed in two ways. First, in some experiments, the high Na calibration buffer contained 30 instead of 90 mM Na+. For these experiments, we converted ratios to
[Na+]i values using a formula slightly different from our standard one, exploiting data from multipoint calibration experiments
(see RESULTS), which included both 30 and 90 mM Na+ data.
This difference in calibration did not lead to appreciable differences in calculated [Na+]i values in the range between 0 and 50 mM (see Fig. 2, below), which includes virtually all of our data.
A second difference was that, in some early experiments, the calibration buffers contained gramicidin and nigericin, but not monensin. We later found that, although the two- and three-ionophore calibrations yielded the same I340/I380 ratios when [Na+]o was 0 mM, the two-ionophore method yielded lower ratios than the three-ionophore method when [Na+]o was 30-90 mM. This result suggested to us that gramicidin plus nigericin did not truly equilibrate [Na+]i with [Na+]o. As shown in RESULTS, however, our flame-photometry data confirmed that the three-ionophore method does indeed equilibrate [Na+]i with [Na+]o. Therefore, we performed additional experiments in which we sequentially perfused the cells with calibration buffer containing 90 mM Na+ and two ionophores, and then calibration buffer containing 90 mM Na+ and all three ionophores. We found that the addition of the third ionophore caused I340 and I380 to decrease by 4 and 7%, respectively. These results allowed us to correct I340 and I380 data in experiments in which we had calibrated with only two ionophores, thereby yielding accurate [Na+]i values.
As a confirmation that the above correction procedures were valid, we examined the ouabain-insensitive Na+ efflux (i.e., Na+ leak) in the various Jewell-Lingrel cell lines. Indeed, our corrected data were similar, regardless of whether the calibration buffer contained 30 or 90 mM Na+, or two or three ionophores.
Determination of [Na+]i by Flame Photometry
To independently validate our method for converting SBFI I340/I380 ratios to [Na+]i values, we used flame photometry to measure [Na+]i. Untransfected HeLa cells were grown in six-well tissue plates until 80-100% confluent. Three wells of each six-well plate were used for measuring extracellular and intracellular volume (ECFV, ICFV), with the other three wells being used for flame photometry measurements of total Na. All cells were incubated in calibration buffers containing gramicidin, nigericin, and monensin, and varying [Na+] as noted above. For the ECFV and ICFV determinations, the solutions also contained 14C-inulin and 3H-H2O (Amersham Corp., Arlington Heights, IL).
At the start of each of these experiments, we aspirated the culture medium and rinsed each well twice with 1 ml of the calibration buffer (prewarmed to 37°C) containing the selected [Na+]
(0-90 mM). After aspirating the second of these 1-ml rinses, we
added 1 ml of the selected calibration buffer to each well and
placed the six-well plates in a sealed vessel containing a tray of
buffer to minimize evaporation. We placed this vessel in a 37°C
incubator for 1-2 h. We next took samples of the solution (i.e.,
ECF) in each well for subsequent scintillation counting. Next, we
rinsed each well twice with 1 ml ice-cold choline chloride (160 mM) to minimize contamination by the original calibration buffer.
Then, we added 0.5 ml lysis solution (Triton X-100, 0.5%) to
each well and allowed cell lysis to proceed overnight with gentle
shaking. We then scraped up and suspended any material adherent to the well, and aspirated this suspension. We rinsed the well
with an additional 0.5 ml lysis solution, which we combined with
the aspirate and centrifuged for 5 min at 15,000 rpm, discarding the pellet (Munzer et al., 1994).
We placed aliquots of samples containing isotopes in scintillation vials and counted them in a Tri-Carb 1600 TR (Packard Instrument Co., Inc., Downers Grove, IL). We separated counts for 14C and 3H using a channels-ratio method. We diluted the nonradioactive lysate samples (as needed), and measured [Na+] in a flame photometer (400; Corning Glass Works, Corning, NY). We also assayed by flame photometry buffer samples taken from each well at the conclusion of the incubation, as well as samples of unused calibration buffer, lysis solution, and ultrapure water in duplicate or triplicate.
In calculating [Na+]i, we assumed that the cell lysate represents ICF, contaminated with some ECF. Because 14C-inulin distributes only in the ECF, the ECFV of the lysate is equal to the 14C-cpm in the cell extract divided by the 14C-cpm/µl of buffer sample. Because 3H-H2O labels all the water in the sample, the corresponding quotient for 3H-cpm represents ECFV + ICFV. The [Na+] of the ECF, as well as the Na+ content (in micromoles) of the cell lysate, are known from flame photometry. Combining this information with the known ECF and ICF volumes allows calculation of the [Na+] of the ICF.
Immunoblotting
We prepared crude microsomes from cells grown to confluence
as previously described (Zahler et al., 1996). The specific activity of Na,K-ATPase in such preparations from untransfected HeLa
cells was ~24 µmol Pi/mg protein per h. We loaded equal
amounts (10-30 µg) of microsomal protein on 7% SDS polyacrylamide gels, performed electrophoresis, and transferred the proteins electrically to Immobilon-P membranes (Millipore Corp.,
Bedford, MA) as previously described (Zahler et al., 1996
). We
performed immunoblots using a 1:500 dilution of a monoclonal
antibody (anti-LEAVE) raised to a pentapeptide (KNCLVKNLEAVE) common to the
1,
2 and
3 isoforms (Pressley, 1992
)
(kind gift of Dr. T. Pressley). We quantitated the results by including titrated amounts of SY5Y microsomal protein on each
blot, measuring band intensity using densitometric scanning, and deriving from this a region where band intensity was a linear function of the amount of protein loaded (Lucchesi and Sweadner, 1991
).
Data Analysis
Results are displayed as mean±SEM. Curve fits were performed
using DeltaGraph 4.0 and Systat 5.1 for Macintosh. We used two approaches for analyzing data for Na+ dependence of pump flux.
First, data were fitted by the highly cooperative model (Garay et
al., 1973; Jewell and Lingrel, 1991)
![]() |
(1) |
where Km = (K)1/3 (Jewell and Lingrel, 1991
), and the exponent
is three because there are three Na+ binding sites. This method is
has been used by previous investigators studying the Na+ dependence of pump flux in transfected cells (Munzer et al., 1994
).
The second approach, feasible because our data had high resolution on the [Na+]i axis, was to fit to a cooperative model with variable Hill coefficient:
![]() |
(2) |
where b is the Hill coefficient.
We also wished to determine statistically whether the effluxes
in the presence of different ouabain concentrations were significantly different. Because the efflux data are heteroscedastic (standard errors are not random, but increase as [Na+]i increases; see
Fig. 5), they are not well suited for evaluation by standard regression methods. Accordingly, we logarithmically transformed the
data; the log effluxes were close to a linear function of [Na+]i,
with uniform standard errors. We tested for equality of the slopes
and intercepts of these linear regression lines. Finally, we tested
for interisoform differences in the affinity constants for [Na+]i
derived from the fitted curves using chi-square analysis and the
asymptotic standard errors of estimate of the affinity constants.
Validation of Ionophore Technique Used for Equilibrating Extracellular and Intracellular [Na+]
As described in METHODS, we incubated untransfected HeLa cells in calibration buffers containing three ionophores (gramicidin, nigericin, and monensin) and various levels of [Na+], with and without 14C-inulin and 3H-H2O. Scintillation counting showed that ECFV and ICFV were approximately equal, increasing the reliability of the calculation of [Na+]i. Combining these data with flame-photometry data for total Na+, we computed [Na+]i. As shown in Fig. 1, incubation with the three ionophores causes intracellular and extracellular [Na+] to become essentially equal over a wide range of [Na+]o values.
Multipoint Calibration of SBFI
Alternately exciting at 340 and 380 nm, while monitoring the emission at 505 nm, we determined the response of the I340/I380 fluorescence-excitation ratio to defined perturbations of [Na+]i using gramicidin, nigericin, and monensin to equilibrate [Na+]i with [Na+]o. A typical multipoint calibration experiment for SBFI in HeLa cells is shown in Fig. 2 A, where the I340/I380 ratio appears to closely track [Na+]o. Accordingly, we combined such multiple experiments and analyzed the resulting I340/I380 vs. [Na+] data, as follows.
Let r ([Na+]i) be the I340/I380 ratio as a function of [Na+]i. Then we define the normalized fluorescence- excitation ratio as
![]() |
(3) |
where r (0) and r (90) are the ratios when [Na+]i is 0 and 90 mM, respectively. This transformation forces r to be zero when [Na+]i is 0, and 1 when [Na+]i is 90 mM. Combining data from eight similar experiments, we then calculated r as a function of [Na+]. The results, shown by the symbols in Fig. 2 B, indicate that the normalized I340/I380 ratio rises monotonically with [Na+]i. The tight confidence intervals (average SEM, 2.6 mM Na) indicate that changes in [Na+]i of 3 mM or less can be routinely detected. Finally, we fitted these data with a right rectangular hyperbola, obtaining the curve shown in Fig. 2 B. Rearranging the equation for this hyperbola, we can compute [Na+]i from the normalized ratio:
![]() |
(4) |
Measurement of Basal [Na+]i
We determined the basal [Na+]i for each of the cell
lines by incubating the cells in standard buffer until the
I340/I380 ratio reached a steady state, and then immediately calibrating. The results are shown in Table II. The
basal [Na+]i value is 18.6 mM in untransfected HeLa
cells, as compared with the value of 13 mM found by
Boardman et al. (1974) in HeLa cells (see DISCUSSION).
The basal [Na+]i for the
3-transfected cells, 50.3 mM,
was significantly greater (P < 0.05 by ANOVA, Scheffé
multiple comparison test) than the corresponding values for the other Jewell-Lingrel cell lines.
Table II. Basal [Na+]i Values in Jewell-Lingrel Cell Lines |
Measuring [Na+]i Dependence of Na-K Pump Flux in Untransfected HeLa Cells
We determined the [Na+]i dependence of the Na-K
pump flux using an approach described by Negulescu
and Machen (1990). We obtained the pump flux as a
function of [Na+]i because the Na-K pump activity is
sensitive to [Na+]i, which it directly modulates. As shown
in Fig. 3, we first Na+-loaded untransfected HeLa cells
(which have only native, human
1 subunits) by incubation in zero-K+ buffer (segment ab). We then removed extracellular Na+, and simultaneously raised the
[K+]o to 5 mM, while monitoring [Na+]i as Na+ left the
cells (bc). When the I340/I380 ratio fell sufficiently, we
performed a two-point calibration as described above
(cd and de). In Fig. 3, ouabain was present during abc at
a concentration (1 µM) high enough to completely inhibit the HeLa cell Na,K-ATPase ( Jewell and Lingrel,
1991
). Thus, the Na+ efflux during bc represents the
Na+ efflux mediated by "leak" pathways as a function of
[Na+]i. In other experiments (not shown), ouabain was
absent during abc so that the Na+ efflux during the
equivalent of bc represented the Na+ efflux mediated
both by the Na-K pump and leak pathways.
We then numerically differentiated the time course
of decrease in [Na+]i (Fig. 3, segment bc) to yield
d[Na+]i/dt as a function of [Na+]i. Fig. 4 A shows the
results of this analysis both for experiments conducted
in the presence () and absence of ouabain (
). As
expected, Na+ efflux was considerably higher in the absence of ouabain than in its presence, and the two
curves are significantly different (P < 0.002). Moreover, the efflux was similar in low (1 µM) and high (4 mM) ouabain concentrations in this cell type (not
shown), as would be expected, given the high ouabain
affinity of the native human
1 in untransfected HeLa
cells. From Fig. 4 A, we obtained the activation curve of
the Na-K pump as a function of [Na+]i by subtracting
the efflux in the presence of ouabain from the efflux in
the absence of ouabain (Fig. 4 B). This activation curve indicates an apparent Km of 12 mM (fit with variable
Hill coefficient, Eq. 2). Others, employing various methods, have obtained similar values: 16 mM in rat brain
synaptosomes (Brodsky and Guidotti, 1990
), and ~10
mM in human erythrocytes (Garay and Garrahan, 1973
).
Although we obtained d[Na+]i/dt data for [Na+]i values larger than 20 mM in the untransfected HeLa cells and the other Jewell-Lingrel cell lines, we found that values obtained immediately after the switch from zero-K+ buffer to zero-Na+ buffer, when [Na+]i was high and falling very rapidly (just after point b in Fig. 3), varied widely from experiment to experiment. A major reason for this variability is the inherent difficulty in computing accurate numerical derivatives (d[Na+]i/ dt) of digital data when [Na+]i is falling rapidly, because the tangent line is fitted to fewer data points. We thus chose to restrict the analysis to [Na+]i ranges in which the d[Na+]i/dt data were reproducible. Therefore, in each experiment, we identified the inflection point in the d[Na+]i/dt versus [Na+]i tracing, and analyzed only data for [Na+]i values lower than the inflection point. This approach had the practical effect of limiting the range of usable data to [Na+]i values from 0 through 30-35 mM.
Measuring [Na+]i Dependence of Flux Attributable to Exogenous Isoforms in Cells Transfected with Ouabain-resistant Pumps
For these experiments, we modified the above protocol to exploit the large difference in ouabain affinities between the native and the transfected Na-K pumps. We loaded cells with Na+ as above, and then exposed them to a 0-Na+/5-mM-K+ buffer (solution 2). In each case, we performed these two maneuvers either in the absence of ouabain, or at low (1 µM) or high (4 mM) ouabain concentrations. We interpreted the corresponding Na+ effluxes, calculated as d[Na+]i/dt, as follows:
In presence of: | Na efflux represents: |
high [ouabain] | Na+ leak |
low [ouabain] | leak + resistant pump rate |
no ouabain | leak + total pump rate |
Here, "resistant" pumps refers to the transfected rat
isoforms, whereas the "sensitive" pumps are the human
1 isoforms endogenously expressed in HeLa cells. In a
generalization of the above approach (Figs. 3 and 4), we
subtracted high from low ouabain data, obtaining the
activation curve of the resistant (i.e., rat) Na-K pump
isoform as a function of [Na+]i. Similarly, subtracting
low from zero ouabain data yields the activation curve
of the sensitive (i.e., human) Na-K pump isoform.
Rat 1
We studied cells transfected with the rat 1 isoform,
which is naturally ouabain resistant, at low and high
ouabain concentrations (Fig. 5 A). The difference between these two Na+ effluxes represents the activity of
the transfected rat pumps. As shown in Fig. 5 B, the
maximal Na+ efflux attributable to the transfected rat
1 pumps was ~0.25 mM/min, considerably less than
that of the native human
1 pumps in untransfected
HeLa cells, ~0.64 mM/min (Fig. 4). This difference may be related to a relatively small number of exogenous pumps. The exogenous rat
1 pumps had apparent Km for [Na+]i of 12 mM, similar to that for the endogenous human
1 pumps in the transfected HeLa
cells (Table III).
Table III. Curve Fits |
Rat 2*
To study the rat 2* isoforms, we again compared experiments in
2*-transfected cells exposed to low and
high ouabain concentrations (Fig. 6 A). The activation
curve for Na+ efflux attributable to this mutated rat isoform had an apparent Km of 22 mM (Fig. 6 B). The
maximal Na+ efflux attributable to the transfected rat
2* isoform was 1.8 mM/min, considerably greater than
that for the transfected rat
1 (Table III).
Rat 3*
We studied cells transfected with 3* (Fig. 7, A and B)
in the same manner as for
1 and
2*. In the case of
the
3*-transfected cells, the computed Vmax for the efflux attributable to
3* was ~2.5 mM/min. More significantly, the activation curve (Fig. 7 B) is substantially
shifted to the right, compared with the other isoforms
studied. Fitting the
3*-flux data in Fig. 7 B using a cooperative model with a Hill coefficient of three (Eq. 1)
(Garay et al., 1973) yielded a Km of ~44 mM. This fit,
however, is problematic for two reasons. First, the efflux is not above zero until [Na+]i is above ~16 mM.
Second, although there is a suggestion of saturation at
an [Na+]i of ~40 mM, we were unable to obtain data at
higher [Na+]i values. Studying the same
3*-transfected
cells with a different approach, Munzer et al. (1994)
also noted that the
3* flux did not begin to increase
until [Na+]i was above ~22 mM. Their flux data, plotted as a function of [Na+]i, did not appear to saturate.
Because our approach generated many more data points
than that of Munzer et al. (1994)
, however, we were
able to fit our data using a model with a variable Hill coefficient. The result was a much better fit than the
model with a fixed Hill coefficient, and a Km of ~33 mM.
For the sake of comparison, we replotted the activation curves for the three transfected isoforms on the
same axes (Fig. 8), normalizing the maximal pump flux
for each isoform to unity. The Km values are in the sequence ~12, ~22, and ~33 mM for the rat 1,
2*,
and
3* isoforms, respectively. To assess whether these values represent true interisoform differences in affinity for [Na+]i, we tested the Na+ efflux curves for human
1, rat
2*, and rat
3* (Figs. 4 B, 6 B, and 7 B) against
the corresponding curve for rat
1, using chi-square
analysis for heterogeneity. Rat
2* and rat
3* were significantly different from rat
1 (P values < 0.001 and
0.005, respectively), but human
1 was not significantly
different from rat
1 (P > 0.75). We also computed asymptotic standard errors of estimate for the affinity
constants, and tested whether they were significantly
different. The results were as follows: rat
2* vs. rat
1,
P < 0.005; rat
3* vs. rat
1, P < 0.0005; rat
3* vs. rat
2*, P < 0.0005; human
1 vs. rat
1, not significant.
Human 1 in HeLa Cells Transfected with Rat
3*
In the cells transfected with rat 3*, we also measured
the efflux attributable to the endogenous human
1
isoform. The apparent Km for the human
1 isoform
was 13 mM (Fig. 9 A and Table III), in close agreement
with the Km for human
1 measured in untransfected HeLa cells, 12 mM. The maximal Na+ efflux attributable to the
1 isoform in
3*-transfected cells (
in
Fig. 9 B) was only 56% as great as in the control HeLa
cells (+ in Fig. 9 B), suggesting that transfection of
(mutated)
3 pumps leads to a compensatory decrease
in endogenous
1 pumps. Indeed, in the HeLa cells
transfected with rat
2*, the Na efflux attributable to
the native
1 was unmeasurably low (not shown). As
shown in Fig. 9 C, the Km values were indistinguishable
for human
1 in untransfected HeLa cells, for human
1 in
3*-transfected HeLa cells, and for rat
1.
Estimation of Maximal Turnover Rate per Pump Site
Because of differences in transfection efficiency and,
perhaps, in maximal intrinsic pumping rate, one must
be cautious in comparing the activation curves of the
rat isoforms shown in B of Figs. 6-8. We thus sought
to measure the number of pump sites of each isoform
type in the transfected cells. Although pump-site quantitation is customarily done using 3H-ouabain binding,
this technique is not feasible for rat
1,
2*, and
3*
pumps because they are highly ouabain resistant. Instead,
we measured the total amount of Na-K
-isoform protein using quantitative immunoblotting with anti-LEAVE
(see METHODS), an antibody raised against an epitope
common to the three Na-K pump
isoforms (Pressley, 1992
).
The immunoblots showed a single major band with
an apparent molecular mass of 100 kD, as previously
described (Pressley, 1992). Using densitometric scanning referred to a standard curve on each blot, we
found that the total Na-K pump immunoreactivity in
microsomes from the
2*-transfected HeLa cells was
97% of that in untransfected HeLa cells (n = 3 determinations) and that of
3*-transfected cells was 93% of
that in untransfected HeLa cells (n = 2). The pump-mediated Na+ efflux we measured in
2*-transfected
cells was ~2.8× that in untransfected HeLa cells, and
the efflux in
3*-transfected cells was ~4.5× that in untransfected HeLa cells. If we assume that the antibody
recognizes all pumps equally well, and that an equal fraction of all immunoreactive pumps is at the plasma
membrane in all cell lines, then it would appear that
the transfected pumps have a higher intrinsic turnover
rate than the native pumps. For example, if the activity
per human
1 is the same in untransfected and
3*-transfected HeLa cells, the maximal activity per rat
3* pump would be 8.7-fold greater than that of human
1.
Advantages of Spectrofluorometry for Measuring Na+ Fluxes Mediated by the Na-K Pump
We have demonstrated that the Na+-sensitive dye SBFI
can be used to measure the Na+ flux attributable to
specific Na-K pump isoforms, as a function of [Na+]i,
in intact transfected cells. A special advantage of our
spectrofluorometric approach is that we were able to
study Na-K pump fluxes in a sided preparation with independent control over [Na+]i and [Na+]o. Additional
advantages are that [Na+]i and d[Na+]i/dt were obtained simultaneously, and with good resolution for both [Na+]i and time. Measuring the fluxes at specific
[Na+]i values is crucial because the Na-K pump flux is
steeply [Na+]i dependent, and because many experimental interventions change both pump flux and [Na+]i
simultaneously. To our knowledge, this is the first study
of specific Na-K-pump isoforms in a sided preparation
permitting independent control of [Na+]i and [Na+]o,
the first systematic investigation of pump-mediated Na+
fluxes by means of spectrofluorometry, the first use of
SBFI to study [Na+]i in transfected cells, and the first
validation of an in vivo SBFI calibration using an independent method for measuring [Na+]i.
Summary of Results
Implications of Km values.Our data indicate that the rat
3* isoform has a Km for [Na+]i that is more than threefold greater than that of rat
1. Rat
2* has an intermediate affinity. The Na-K pump ought to be most effective at clamping [Na+]i when the Km of the pump is
close to [Na+]i. The existence of an
3 isoform with
such a high Km might help prevent [Na+]i from rising
excessively in cells subjected to high Na+ influxes (e.g.,
excitable cells during electrical activity). This advantage,
however, would accrue only if the extra expression of
3 increased the total number of Na-K pumps, or if the
3 pumps had a higher turnover number. Alternatively, expression of
3, with its high Km, would cause
the cell to have a high steady state [Na+]i, as we indeed
observed. Such a high [Na+]i might be advantageous
because it would reduce ATP use.
Our data also indicate that the Km values for the endogenous human 1 and transfected rat
1 are almost identical, even though the former is 1,000× more sensitive to
ouabain than the latter, This similarity in Km values is
consistent with the notion that the structural elements of
the pump that determine ouabain binding (Lingrel and
Kuntzweiler, 1994
) can be modified independently of
those that determine the Km for internal Na+.
We also found that transfection of rat 2*
or
3* pumps leads to a compensatory decrease in the
activity of endogenous human
1. These observations
are consistent with previous evidence that the total number of Na-K pump units expressed per cell is tightly regulated (Boardman et al., 1972
; Fambrough et al., 1987
;
Pressley, 1992
).
Earlier Work on Na+ Affinity of Pump Isoforms
To study the Km of the Na-K pump for intracellular Na+
(Km[Na+]i), it would be best to use a "sided" preparation in which one could control [Na+]i and [Na+]o independently, and thus vary the parameter of interest
(i.e., [Na+]i) while leaving other relevant parameters
(e.g., [Na+]o) fixed. Such an approach has previously
been used only to study the native Na-K pumps of erythrocytes (e.g., Garay et al., 1973) and squid axons (e.g.,
Brinley and Mullins, 1967).
Varying [Na+]i while
equalizing it to [Na+]o yields what might be called an
"unsided" Km for Na+, Km[Na+]u. 86Rb fluxes in rat-brain synaptosomes, obtained in the presence of low
and high ouabain concentrations, yielded apparent
Km[Na+]u values of 17 mM for 1 and 49 mM for
2
plus
3 (Brodsky and Guidotti, 1990
), consistent with
our Km[Na+]i data. In contrast, ATPase assays on rat
axolemma, which predominantly have the
3 isoform
(Sweadner, 1985
) and the pineal gland, which is predominantly
3
2 (Shyjian et al., 1990
), yielded Km[Na+]u
values that were lower than for kidney, which is predominantly
1.
Several
groups have studied Na-K pump physiology by transfecting cloned Na-K pump isoforms into mammalian
cells. However, measuring Km[Na+] in such cells is complicated by the presence of the endogenous 1 isoform
in mammalian cell lines. One way of circumventing this problem is to express mammalian
1 in nonmammalian cells that have little endogenous Na-K pump activity, such as Sf-9 insect cells (Blanco et al., 1993
) or yeast
(Horowitz et al., 1990
). Thus, in membranes obtained
from yeast cells transfected with
1
1, the Km[Na+]u
for the Na,K ATPase activity was 9.3 mM, similar to the
Km[Na+]i value we obtained for intact, untransfected
HeLa cells, which also express
1
1 (Eakle et al., 1995
).
Jewell and Lingrel (1991) circumvented the problem of endogenous Na-K pumps by transfecting ouabain-resistant pumps into cells whose native pumps are
ouabain sensitive. Using differential ouabain sensitivity
in crude plasma membranes from such cells, those authors found that the Km[Na+]u for the Na,K-ATPase activities of the transfected rat
1,
2*, and
3* isoforms
were 3.5, 3.3, and 7.8 mM, respectively.3 Although these
values are substantially lower than ours, both studies
conclude that the Km for
3 is greater than that for the other isoforms. The difference in magnitude may reflect the use of an unsided vs. a sided preparation.
Putnam et al. (1994) immunoblotted
the Jewell-Lingrel cell lines with an antibody that cross-reacts with human and rat
1, and also measured Na,K-ATPase activity. They found that transfecting with rat
1 or
2* decreases expression of immunoreactive
1, and that transfecting with rat
1,
2*, or
3* reduces
ATPase activity of endogenous
1. These results are
consistent with our efflux measurements. Putnam et al.
also found that all three transfected lines had about
half the total Na,K ATPase activity of untransfected HeLa cells. This result is at odds with our data, which
show that the total Na-K pump flux was higher in the
2*- and
3*-transfected lines. The discrepancy could
reflect either differences in assay methods (i.e., ATPase
activity vs. flux), or in pump localization (total pumps
vs. those in the plasma membrane).
Munzer et al. (1994) studied the [Na+]i
dependence of the pump-mediated 86Rb+ influx, using
monensin as a tool to change [Na+]i in response to
changes in [Na+]o. Km[Na+]u values derived by Munzer
et al. (1994)
from fitting their data to a cooperative
three-site model are shown in Table IV (column 2). More
recently, considering the competition between intracellular Na+ and K+ for cytoplasmic Na+ binding sites of
the Na,K-ATPase in these transfected cells (Therien et
al., 1996
), this group revised its estimates for Km[Na+]u
(column 3). Our Km[Na+]i values, obtained from the
same Jewell-Lingrel cell lines, are similar to the Km[Na+]u
values published by Therien et al. (1996)
. They concluded that the Km[Na+]u values are in the sequence
1
2* <
3*, whereas our data indicate that the sequence is
1 <
2* <
3*.
Table IV. Comparison between Pump-mediated Na+ Efflux Data in Jewell-Lingrel Cell Lines, as Measured in This Study and in Previous Publications |
It is reassuring that our data and those of Munzer et
al. (1994) are so similar, given the differences in methods: (a) Munzer et al. (1994)
measured fluxes with
86Rb, whereas we used SBFI. (b) Munzer et al. (1994)
made their flux measurements in the presence of monensin, whereas we made ours in the absence of ionophores.4 (c) Munzer et al. (1994)
did not measure [Na+]i
directly, but assumed it to be equal to [Na+]o in their
monensin-treated cells. We measured [Na+]i directly,
in real time. (d) Our sampling technique allowed us to
generate many more ([Na+]i, flux) data pairs along the
[Na+]i activation curve, so that we were justified in fitting our data to a model with an additional parameter
(i.e., variable Hill coefficient, as in Table IV).
Previous Work with SBFI
SBFI has been used to measure [Na+]i in many cell
types (Harootunian et al., 1989; Harrison et al., 1992
;
Ahlemeyer et al., 1992
). Some investigators have incidentally used SBFI to estimate an instantaneous Na+
flux. For example, Kondo et al. (1993)
Na+ loaded cells
of the thin ascending limb of Henle's loop by removing extracellular K+, and then measured the initial rate of
[Na+]i decrease after restoring the K+. Similar experiments were reported by Harootunian et al. (1989)
on
fibroblasts and Borin et al. (1993)
on vascular smooth-muscle cells. Because the above studies were performed
only in the absence of ouabain, it was not possible to
separate the Na-K pump flux from Na+ leak. Moreover,
they measured the flux at a single [Na+]i, but did not
attempt to measure the dependence of the Na+ efflux
on [Na+]i.
SBFI Calibration Procedure
To increase the accuracy of our measurements, we calibrated the SBFI at two [Na+]o values in each experiment. Several ionophore combinations have been used
to calibrate SBFI in vivo; however, we know of no example in which an independent measurement of [Na+]i
was used to verify that any of these combinations truly
equalizes [Na+]i and [Na+]o. Using untransfected HeLa
cells, we systematically investigated the effects of adding gramicidin, nigericin, and monensin, stepwise in all
possible sequences, to a calibration buffer containing 30-90 mM Na+. We found that the SBFI fluorescence-
excitation ratio consistently increased when any one of
the ionophores was added, even when the solution already contained one or two other ionophores (not shown). Adding ionophores had little effect, on the
other hand, when the calibration buffer was Na-free.
These results indicate that no two of the three ionophores were adequate to totally permeabilize the cells
to Na+. We then demonstrated, using flame photometry, that the combination of gramicidin, nigericin, and
monensin (Harootunian et al., 1989; Tepel et al., 1994
)
does in fact equalize [Na+]i and [Na+]o over a wide
range of [Na+]o in HeLa cells.
Potential Limitations
Generalizability of conclusions.Kinetic parameters of the
Na-K pump isoforms may depend, in part, on the tissue in which they are expressed (Therien et al., 1996
).
Cell-specific variations could arise from differences in
such parameters as posttranslational modification, intracellular ionic composition, and membrane-lipid
composition. Another variable is
-subunit isoforms.
HeLa cells contain only the
1 isoform; it is possible
that the kinetic properties of a specific
could be altered by pairing it instead with
2 or
3 ( Jaisser et al., 1992
; Eakle et al., 1992
; Cameron et al., 1994
; Eakle et
al., 1995
).
In our flux analysis, we
assume that Na+ buffering is negligible, as has been
verified in gastric parietal cells (Negulescu and Machen, 1990). Previous studies have sometimes revealed
differences between intracellular free Na+ and total
cell Na+ content (Negulescu and Machen, 1990
), or differences in [Na+]i among intracellular compartments.
Borin et al. (1993)
, however, using digital imaging microscopy of SBFI-loaded smooth muscle cells, observed
that, although the 340/380 fluorescence ratio was not
uniform throughout the cell, the [Na+]i calculated from
an in situ (i.e., intracellular) calibration was uniform.
Original version received 30 January 1997 and accepted version received 9 May 1997.
Address correspondence to Raphael Zahler, MD, PhD, Section of Cardiology/Fitkin 3, P.O. Box 208017, Yale University School of Medicine, 333 Cedar St., New Haven CT 06510. Fax: 203-785-7144; E-mail: raphael.zahler{at}yale.edu
2 Care was taken to thoroughly rinse the perfusion lines with 70% EtOH and distilled water between experiments so as to remove traces of ouabain and ionophores.We thank Wei Sun, Mark Lufburrow, Eugene Bang, and Dong-Hong Zhang for excellent technical assistance. We are very grateful to Drs. Jerry Lingrel and Beth Jewell-Motz for giving us the transfected HeLa cells. The anti-LEAVE antibody was a kind gift of Dr. T. Pressley. We also gratefully acknowledge helpful comments from Drs. Ed Benz, Satish Singh, Mark Bevensee, Joseph Hoffman, and Clive Orchard.
Supported by National Science Foundation grant IBN-9421171, National Institutes of Health Program Project Grant DK-17433, The Patrick and Catherine Weldon Donaghue Foundation, and The Connecticut Affiliate of The American Heart Association.
SBFI, sodium-binding benzofuran isophthalate.