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Sodium kinetics of Na,K-ATPase alpha isoforms in intact transfected HeLa cells.

Zahler R, Zhang ZT, Manor M, Boron WF - J. Gen. Physiol. (1997)

Bottom Line: We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1.The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps.We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA. raphael.zahler@yale.edu

ABSTRACT
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 alpha isoforms of the pump, alpha1 is ubiquitous, alpha2 is predominant in skeletal muscle, and alpha3 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 alpha subunits were highly ouabain resistant: the alpha1 isoform is naturally resistant, whereas the alpha2 and alpha3 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 microM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1. The alpha2 and alpha3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing alpha3 had a high resting [Na+]i. The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3. The alpha3 isoform may be suited for handling large Na+ loads in electrically active cells.

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Na+ efflux in untransfected HeLa cells. (A) Dependence of rate of [Na+]i decrease on [Na+]i, computed from experiments similar to that shown in Fig. 3. Data were obtained either in  the absence of ouabain (▪, n = 7), or with 1 μM or 1 mM ouabain  added to the zero-K+ and zero-Na+ buffers (•, n = 9). d[Na+]i/dt  was calculated from the time course of [Na+]i decline in the presence of zero-Na+ buffer (Fig. 3, segment bc). These calculations  were performed at [Na+]i intervals of 1 mM. Statistical analysis (see  methods) shows that the two curves are significantly different (P <  0.002). (B) [Na+]i dependence of the Na+ efflux attributable to  the Na-K pump. The d[Na+]i/dt in the presence of ouabain is interpreted as the Na+ leak, whereas the d[Na+]i/dt in the absence  of ouabain is interpreted as sum of the leak and the flux mediated  by the Na-K pump. Thus the difference, shown here, represents  the pump-mediated efflux. Data were fit with a cooperative model  with variable Hill coefficient (Eq. 2).
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Figure 4: Na+ efflux in untransfected HeLa cells. (A) Dependence of rate of [Na+]i decrease on [Na+]i, computed from experiments similar to that shown in Fig. 3. Data were obtained either in the absence of ouabain (▪, n = 7), or with 1 μM or 1 mM ouabain added to the zero-K+ and zero-Na+ buffers (•, n = 9). d[Na+]i/dt was calculated from the time course of [Na+]i decline in the presence of zero-Na+ buffer (Fig. 3, segment bc). These calculations were performed at [Na+]i intervals of 1 mM. Statistical analysis (see methods) shows that the two curves are significantly different (P < 0.002). (B) [Na+]i dependence of the Na+ efflux attributable to the Na-K pump. The d[Na+]i/dt in the presence of ouabain is interpreted as the Na+ leak, whereas the d[Na+]i/dt in the absence of ouabain is interpreted as sum of the leak and the flux mediated by the Na-K pump. Thus the difference, shown here, represents the pump-mediated efflux. Data were fit with a cooperative model with variable Hill coefficient (Eq. 2).

Mentions: 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 α1 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).


Sodium kinetics of Na,K-ATPase alpha isoforms in intact transfected HeLa cells.

Zahler R, Zhang ZT, Manor M, Boron WF - J. Gen. Physiol. (1997)

Na+ efflux in untransfected HeLa cells. (A) Dependence of rate of [Na+]i decrease on [Na+]i, computed from experiments similar to that shown in Fig. 3. Data were obtained either in  the absence of ouabain (▪, n = 7), or with 1 μM or 1 mM ouabain  added to the zero-K+ and zero-Na+ buffers (•, n = 9). d[Na+]i/dt  was calculated from the time course of [Na+]i decline in the presence of zero-Na+ buffer (Fig. 3, segment bc). These calculations  were performed at [Na+]i intervals of 1 mM. Statistical analysis (see  methods) shows that the two curves are significantly different (P <  0.002). (B) [Na+]i dependence of the Na+ efflux attributable to  the Na-K pump. The d[Na+]i/dt in the presence of ouabain is interpreted as the Na+ leak, whereas the d[Na+]i/dt in the absence  of ouabain is interpreted as sum of the leak and the flux mediated  by the Na-K pump. Thus the difference, shown here, represents  the pump-mediated efflux. Data were fit with a cooperative model  with variable Hill coefficient (Eq. 2).
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Figure 4: Na+ efflux in untransfected HeLa cells. (A) Dependence of rate of [Na+]i decrease on [Na+]i, computed from experiments similar to that shown in Fig. 3. Data were obtained either in the absence of ouabain (▪, n = 7), or with 1 μM or 1 mM ouabain added to the zero-K+ and zero-Na+ buffers (•, n = 9). d[Na+]i/dt was calculated from the time course of [Na+]i decline in the presence of zero-Na+ buffer (Fig. 3, segment bc). These calculations were performed at [Na+]i intervals of 1 mM. Statistical analysis (see methods) shows that the two curves are significantly different (P < 0.002). (B) [Na+]i dependence of the Na+ efflux attributable to the Na-K pump. The d[Na+]i/dt in the presence of ouabain is interpreted as the Na+ leak, whereas the d[Na+]i/dt in the absence of ouabain is interpreted as sum of the leak and the flux mediated by the Na-K pump. Thus the difference, shown here, represents the pump-mediated efflux. Data were fit with a cooperative model with variable Hill coefficient (Eq. 2).
Mentions: 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 α1 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).

Bottom Line: We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1.The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps.We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA. raphael.zahler@yale.edu

ABSTRACT
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 alpha isoforms of the pump, alpha1 is ubiquitous, alpha2 is predominant in skeletal muscle, and alpha3 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 alpha subunits were highly ouabain resistant: the alpha1 isoform is naturally resistant, whereas the alpha2 and alpha3 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 microM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1. The alpha2 and alpha3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing alpha3 had a high resting [Na+]i. The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3. The alpha3 isoform may be suited for handling large Na+ loads in electrically active cells.

Show MeSH
Related in: MedlinePlus