INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

EXTRACELLULAR FLUID (ECF) and intracellular fluid (ICF) K⁺ levels are tightly controlled in mammals because their ratio is the principal determinant of cell membrane potentials. When whole body K⁺ output exceeds K⁺ input over time, the plasma K⁺ level falls, a condition known as hypokalemia (40). This condition can occur during loop diuretic treatment, which increases K⁺ excretion, or if K⁺ intake is restricted, such as during prolonged fasting (40). In excitable tissues such as heart tissues, hypokalemia-induced disturbances in membrane potential can lead to life-threatening cardiac arrhythmias (10). K⁺ balance is maintained by the interplay of two key organ systems: the kidneys, which can secrete or actively reabsorb K⁺, and skeletal muscle, the major K⁺ reservoir, which can acutely control the transport of K⁺ between the ECF and the ICF (33, 40). Extracellular K⁺ loss is buffered by the transfer of muscle cell K⁺ to the ECF. This loss is likely mediated by the accompanying decrease in Na⁺ pump (Na⁺-K⁺-ATPase) levels, which would decrease active K⁺ transport from ECF to ICF (5, 22, 36).

The Na⁺ pump is a ubiquitous integral membrane P-type ion pump that pumps Na⁺ out of the cell and K⁺ into the cell, a process driven by the hydrolysis of ATP (27). It is an heteromer composed of a catalytically active -subunit (M_r  112,000) and a glycosylated -subunit (M_r  35,000), and recent evidence suggests that it is a tetramer (44). Multiple - and -subunit isoforms are expressed in a tissue-specific manner (13, 29, 41, 42). In skeletal muscles, Na⁺ pump isoforms are expressed in a muscle fiber type-specific manner (19, 43). At the phenotypic fiber type extremes, slow-twitch oxidative soleus expresses 1-, 2-, and 1-isoforms as 11 and 21, whereas fast-twitch glycolytic white gastrocnemius (WG) expresses 1-, 2-, and 2-isoforms as 12 and 22 (43). Background information on the proportion of 2- to 1-type Na⁺ pumps in muscle is limited to two studies. For rat diaphragm membranes the fraction of 2 was estimated at between 30 (in hypothyroids) and 65% (in hyperthyroids) by a backdoor phosphorylation assay that measures pumps undergoing a reaction cycle (15). In rat red (oxidative) skeletal muscle subjected to subcellular fractionation on sucrose gradients, the ratio of 2 to 1 ranged from 1.6 (60% 2) in surface membranes to 7 (87% 2) in intracellular membranes. The ratio was calculated by using ouabain binding per milligram of protein to quantitate 2, and the immunoblot signal was scaled against the signal from a recombinant fragment of 1 per milligram of protein to measure 1 (23). In comparison, the proportion of 2 to total  mRNA in skeletal muscle has been reported as being between 70 and 80% (13, 37). The ratio of total 2 to total 1 in control muscles, either oxidative or glycolytic, has not been previously determined.

We previously reported that, when rats are fed a K⁺-deficient diet for 10 days, Na⁺ pump protein changes are isoform and muscle fiber type specific (43): in fast-twitch glycolytic WG, 2 and 2 protein levels decrease to only 6 and 30% of control, respectively, whereas in slow-twitch oxidative soleus 2 decreased to 45% of control and 1 did not decrease significantly. The 1 protein pool size for either muscle did not change. Despite the substantially greater decrease in 2 and 2 in WG than in soleus, the fall in whole-muscle tissue K⁺ level was ~20% after 10 days of K⁺ restriction in both muscles. These findings provoked questions about the muscle-specific mechanisms of cell K⁺ loss. The first aim of this study was to learn whether the decrease in 2 abundance is linked to the loss of cell K⁺ by comparing the time courses of the two parameters at early time points in the response. The second aim was to measure both protein and mRNA levels of Na⁺ pump subunits over the time course of K⁺ depletion to understand the molecular mechanisms responsible for the decrease in expression in different muscle types. The third aim was to assess the ratio of 2- to 1-isoforms because it will affect the impact of 2-specific regulation. The present study demonstrates that changes in 2 protein levels occur early enough to account for the loss of cell K⁺ in both muscle types, that the decrease in 2 protein is not secondary to a decrease in 2 mRNA, and that the calculated percentage of 2-type pumps in control muscles ranges from 40% in soleus to 55% in WG.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals and diets. Male Sprague-Dawley rats, ~8 wk of age (250-300 g), were placed on a K⁺-deficient diet (TD 88239; Harlan Teklad, Madison, WI) for 2, 5, or 10 days and were paired to controls fed a comparable diet with K⁺ restored (TD 88238; Harlan Teklad). Rats were anesthetized with 0.2 ml pentobarbital sodium/100 g body wt. Soleus and WG hindlimb muscles were removed, frozen in liquid nitrogen, and stored at 80°C pending analyses. Blood samples were taken from the abdominal aorta, and the serum was separated, frozen, and stored at 20°C pending analyses.

Serum and intracellular electrolytes. Serum and muscle cell K⁺ and Na⁺ concentrations ([K⁺] and [Na⁺]) were measured by flame photometry. Muscles were thawed, blotted lightly to remove adherent fluid, and homogenized in 0.3 M trichloroacetic acid [TCA; 1:50 (wt/vol)] for 5 min with a Tissuemizer homogenizer and then centrifuged at 2,500 rpm for 20 min to remove cell debris. The K⁺ and Na⁺ contents of the muscle TCA extracts and serum samples were measured with an FLM 3 flame photometer (Radiometer, Copenhagen, Denmark), with lithium as the internal standard (21). The total muscle [K⁺] and [Na⁺] were corrected for the levels of these cations in the extracellular space.

Na⁺-K⁺-ATPase - and -subunit immunoreactivities. These immunoreactivities were determined as previously described (43). In brief, skeletal muscle was homogenized 1:20 (wt/vol) in 5% sorbitol, 25 mM histidine-imidazole (pH 7.4), 0.5 mM EDTA disodium, and proteolytic enzyme inhibitors [0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, and 1 mM 4-aminobenzamidine dichloride (pABAD)] with a Polytron homogenizer. To facilitate detection of the -subunit, sugar residues were removed from -subunits with PNGase F as previously described (43). A constant amount of homogenate protein (100 µg for -subunit analysis, 50 µg for -subunit analysis) was resolved by SDS-PAGE and blotted onto Immobilon-P membranes (Millipore, Bedford, MA). Blots were incubated overnight with one of the following antibodies: McB2, a monoclonal antibody specific for 2 (45), generously provided by K. Sweadner (Harvard Medical School); anti-1 FP (1:500), a polyclonal antibody against 1 (43); SpET b2 (1:2,000), a polyclonal antibody against human 2 (14), generously provided by P. Martin-Vasallo (Universidad de La Laguna); and RNT 3 (1:2,000), a polyclonal antibody against rat 3 (4), provided by K. Sweadner. Blots probed with monoclonal McB2 were incubated for 2 h with rabbit anti-mouse IgG secondary antibody (Calbiochem, La Jolla, CA; 1:2,000). Antibody-antigen complexes labeled with ¹²⁵I-protein A were visualized by autoradiography as described previously (28), and linearity was verified by assaying samples at multiple concentrations.

Na⁺ pump enzymatic activity. Na⁺ pump activity in crude muscle homogenates was estimated by the K⁺-dependent p-nitrophenylphosphatase (K⁺-pNPPase) reaction (35), as recommended by Kjeldsen et al. (22) because levels of ouabain-sensitive Na⁺-K⁺-ATPase in skeletal muscle homogenate cannot be reliably determined, presumably because of the ouabain resistance of 1 in rats, the low abundance of Na⁺ pumps, and/or the high level of other ATPases in skeletal muscle. In brief, crude homogenates were freeze-thawed three times to permeabilize membranes, and 60 µg of homogenate protein were added to 500-µl sets of K⁺-pNPPase assay mixtures containing 100 mM KCl or 100 mM NaCl. The [Na⁺] carried over from the homogenization buffer was <0.5 mM. Activity is reported as micromoles of phosphate per milligram of protein per hour.

Na⁺-K⁺-ATPase  and  mRNA analysis. Total RNA was isolated from rat skeletal muscle as described by Chomczynski and Sacchi (8) with Tri Reagent (Molecular Reasearch Center, Cincinnati, OH) according to the manufacturer's protocol. RNA concentrations were determined by measuring the optical density at 260 nm (OD₂₆₀), and purity was estimated by determining the OD₂₆₀/OD₂₈₀ ratio. Total RNA was assayed by Northern analysis, as previously described (17, 32), on a NitroPure nitrocellulose transfer membrane (Micron Separations, Westborough, MA). Immobilized RNA was hybridized with isoform-specific restriction endonuclease fragments (~300 bp) prepared from either 1, 2, or 1 clones as described by Orlowski and Lingrel (37) or from the rat 2 cDNA clone provided by P. Martin-Vasallo (30). The - and -cDNA probes were labeled to similar specific activities with [³²P]dCTP probes by using a multiprimer DNA-labeling technique (12). Blots were washed three times in 2× SSC (0.3 M NaCl-0.03 M sodium citrate, pH 7.0) with 0.05% SDS at room temperature for 10 min each and then twice for 20 min each in 0.1× SSC with 0.1% SDS at 55°C. Autoradiograms of the blots were quantified by scanning densitometry.

Quantitation. Autoradiograms were quantitated by scanning with a GS670 imaging densitometer (Bio-Rad, Hercules, CA) and dedicated software. All data are expressed as means ± SE, normalized to the mean value at day 0, defined as 1. Significance was assessed by the two-tailed Student's t-test, and differences were considered significant at P < 0.05. The fractions of 1- and 2-isoforms in soleus and WG muscles were estimated as described in RESULTS. Parameter identifications were performed with MLAB software (Civilized Software, Bethesda, MD), implemented on an IBM-compatible computer, which uses a Marquardt-Levenberg iterative least-squares algorithm. Again, data are reported as fractions of total pumps (defined as 1.0) ± SE.

Materials. Chemicals were reagent-grade, spectroquality, or electrophoresis purity reagents. SDS-PAGE reagents were from Bio-Rad. Leupeptin, PMSF, pABAD, and SDS-PAGE molecular weight standards were from Sigma Chemicals (St. Louis, MO). PNGase F (N-glycanase) was from Genzyme Corporation (Cambridge, MA). ¹²⁵I-protein A and [³²P]dCTP were from ICN (Costa Mesa, CA). L-[³H]glucose was from DuPont NEN (Boston, MA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Time course of change in extracellular [K⁺] during K⁺ deprivation. In 8-wk-old rats placed on a K⁺-deficient diet, serum [K⁺] fell significantly from a control value of 4.6 ± 0.08 to 3.7 ± 0.09 mM by day 2 and continued to fall to 3.3 ± 0.2 and 2.9 ± 0.2 mM by days 5 and 10, respectively (Fig. 1). The fall in serum [K⁺] during the first 2 days (0.9 mM) is greater than or equal to the fall during the subsequent 8 days (0.8 mM), the time when renal and muscle adjustments to hypokalemia come into play (26, 48). Serum [Na⁺] did not change throughout the course of the study. The fall in serum [K⁺] is consistent with previous reports (22, 36) and demonstrates that K⁺ output exceeds input in this K⁺ deprivation model.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Time course of change in extracellular (serum) K+ and Na+ concentrations ([K+] and [Na+]) during K+ restriction. Measurements were made by flame photometry, as described in MATERIALS AND METHODS. Results are means ± SE. * P < 0.05 (significant result). Sample sizes for each time point are in parentheses above abscissa.

Time course of change in Na⁺-K⁺-ATPase isoform abundance vs. cell [K⁺] in soleus and WG muscles during K⁺ deprivation. To determine whether changes in Na⁺ pump expression precede the decrease in cell [K⁺] and to assess the relative contributions of distinct muscles in the adaptive responses to hypokalemia, two muscles at phenotypic extremes were chosen for study: soleus, with 87% slow-twitch oxidative fibers and some fast-twitch glycolytic-oxidative fibers, and WG with 84% fast-twitch glycolytic fibers and some fast-twitch oxidative fibers (2, 3).

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Detection of Na+-K+-ATPase - and -isoforms in muscles from control (C) and K+-deprived (K) rats at 2, 5, and 10 days (2d, 5d, and 10d, respectively). Representative autoradiograms of immunoblots of homogenate samples from soleus (A) and white gastrocnemius (WG; B) (100 µg for 2 and 50 µg for 1 or 2) are shown. The 1- and 2-subunit immunoreactivities were measured after removal of sugar residues with N-glycanase, as described in MATERIALS AND METHODS. Samples from control and K+-restricted animals at a time point were run on the same gel and processed identically. Thus comparisons should be made at a given time point between results for control and K+-deprived rats (i.e., horizontally), not between results for control or K+-deprived rats at different times (i.e., vertically), because of variable incubation and exposure times of different autoradiograms. The antibody-antigen complexes were detected with 125I-protein A.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Time courses of change in Na+ pump subunits and cell cation concentrations during K+-restricted diet. Na+-K+-ATPase 2, 1, and 2 immunoreactivities and intracellular [K+] and [Na+] in soleus (A) and WG muscles (B) are shown. A, top, and B, top: summaries of subunit immunoreactivities, which were assessed by scanning densitometry as described in MATERIALS AND METHODS. Data are means ± SE and are expressed relative to mean control values, defined as 1. * P <0.05 (significant result). Sample sizes: 5 for days 0, 2, and 5; 7 for day 10. A, bottom, and B, bottom: summaries of intracellular [K+] and [Na+], determined by flame photometry and corrected for extracellular space as described in MATERIALS AND METHODS. Sample size was 5 for all data points. Data are means ± SE. * P < 0.05 (significant result).

Estimating the proportion of the Na⁺-K⁺-ATPase 2-isoform in soleus and WG. It has been difficult to directly assess the ratio of 1- to 2-type Na⁺ pumps in muscle. Although absolute pool sizes of 2 vs. 1 protein subunits cannot be determined directly with antibody probes, muscle-specific changes in 2 vs. 1 or 2 abundance during K⁺ deprivation can be employed to calculate the ratios of 2 to 1. The calculations depend on three assumptions. First, 1 and 2 proteins are assumed to combine with only 1 in soleus and only 2 in WG in a 1:1 stoichiometry. Indeed, recent evidence suggests that Na⁺ pumps may exist as tetramers with a 1:1 -to- stoichiometry in mammalian cells (44). Second, it is assumed that there are only negligible pools of uncomplexed - or -subunits, a difficult assumption to test in this system but supported by evidence that - and -subunits form complexes as heteromers before leaving the endoplasmic reticulum and that increasing the synthesis of one subunit increases the stability of the other, implying that uncomplexed subunits are less stable (discussed in Ref. 25). Third, the abundance or pool size is a linear function of the autoradiographic signal, a fact established in previous investigations (6, 28, 46). It follows from these assumptions that the total number of -subunits is equal to the total number of -subunits (Eq. 1), the total number of -subunits is equal to the sum of 1- and 2-subunits (Eq. 2), and thus that the total number of  pumps is equal to the sum of 1 and 2 pumps (Eq. 3)

(1)

(2)

(3)

The number of - or -subunits is equal to the measured immunoblot autoradiographic signal (S), expressed as fraction of control, times a constant (C) related to antibody efficiency (Eqs. 4a-4c)

(4a)

(4b)

(4c)

Thus Eq. 3 becomes Dividing by C Defining A as (C₁/C) and B as (C₂/C)

(5)

Because the 1 signal does not change during hypokalemia, S₁ = 1 (control defined as 1) and

(6)

A and B are estimated by regression analysis, and relative proportions of 1 and 2 pumps are calculated for any time point from the immunoblot signals as follows

(7)

(8)

The fractions of pumps that are 1 and 2 were calculated by simple linear regression (Eq. 6) and are summarized in Table 1. Similar values were obtained by employing multiple regression analysis (Eq. 5) using 1 signals at either 0 or 10 days. In the control state, 2 is calculated to make up 0.4 ± 0.12 of total  in soleus (0.6 ± 0.12 1) and 0.55 ± 0.07 of total  in WG (0.45 ± 0.07 1). After 10 days of K⁺ deprivation, 1 is estimated at 0.76 ± 0.09 of the total pumps in soleus and 0.92 ± 0.09 of the total pumps in WG.

Na⁺ pump enzymatic activity. Na⁺-K⁺-ATPase activity in muscle homogenates of soleus and WG was measured via the K⁺-pNPPase reaction as micromoles of P_i per milligram of protein per hour. Activities in soleus and WG were 0.034 ± 0.004 and 0.033 ± 0.002 µmol P_i · mg protein¹ · h¹, respectively (n = 6 for each). After 10 days of a K⁺-restricted diet, activity was 0.028 ± 0.002 µmol P_i · mg protein¹ · h¹ in soleus, which was not significantly different from control, and 0.026 ± 0.002 µmol P_i · mg protein¹ · h¹ (P < 0.05) in WG (n = 6). This 19% decrease in WG activity falls short of the 70% decrease predicted from the decrease in the 2 pool in this tissue. In comparison, in soleus there is only a 20% fall in 1, which comes close to the measured change in total activity. We conclude that enzymatic activity is not a direct function of abundance. For example, the change in cell Na⁺ that ensues during K⁺ deprivation may provoke a modification of the 1, in WG which persists through homogenization, that changes activity per pump.

Na⁺-K⁺-ATPase mRNA expression in soleus and WG muscles during K⁺ deprivation. Considering the 35-40% decreases in 2 in both muscles after 2 days of K⁺ deprivation and the >90% decrease in WG 2 after 10 days, we tested the hypothesis that the response was driven by decreases in 2 mRNA in both muscles. Na⁺-K⁺-ATPase 2- and -isoform mRNA levels in both muscles were measured after 0, 2, 5, and 10 days of K⁺ deprivation. Northern blot results indicate no change in 2-, 1-, or 2-isoform mRNA expression in either muscle during 10 days of K⁺ deprivation (Fig. 4, A and B). Although distinct bands at the appropriate sizes were observed by this analysis, the quality of the RNA was compromised by the time it takes to dissect the muscles. To circumvent this problem and to reexamine our previous report of a decrease in whole hindlimb 2 mRNA in rats deprived of K⁺ (5), we repeated the mRNA analysis in whole hindlimb muscle isolated as quickly as possible. The results confirmed the findings for the individual muscles: 2, 2 (Fig. 4C), and 1 (not shown) mRNA levels in whole hindlimb did not change during 10 days of K⁺ deprivation.

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Northern hybridization analysis of Na+-K+-ATPase 2, 1, and 2 mRNA during K+-restricted diet for 2 or 10 days. Total RNA (15 µg/lane) was fractionated on formaldehyde agarose gels, blotted onto nitrocellulose, and hybridized with 32P-labeled 2- or -isoform-specific probes as described in MATERIALS AND METHODS. Representative autoradiograms of soleus (A), WG (B), and whole hindlimb (C) Northern blots are shown. Two 2 mRNA species (5.3 and 3.4 kb), 2 1 mRNA species (2.7 and 2.3 kb), and 1 2 mRNA species (2.8 kb) are shown. In C, whole hindlimb control (C) vs. 2-day and control vs. 10-day results are from 2 separate autoradiograms.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The existence of Na⁺ pump isoforms suggests the potential for isoform-specific function, expression, and regulation (31). The need to shift K⁺ from intracellular stores to the ECF is satisfied by tissue-specific expression and regulation of the Na⁺-K⁺-ATPase 2-isoform in skeletal muscle. This report demonstrates that changes in the 2-, not the 1-, isoform occur early enough during K⁺ deprivation to drive the shift of cell K⁺ to the ECF, that there are distinct patterns of regulation in slow-twitch oxidative vs. fast-twitch glycolytic muscles, and that the regulation of 2 occurs by posttranscriptional regulation. As muscle cell [K⁺] falls during K⁺ deprivation, cell [Na⁺] reciprocally increases to as high as 37 mM in WG, which is a classical stimulus for increasing Na⁺ pump activity in most tissues (11). However, skeletal muscle has an altruistic response to elevated cell [Na⁺]: Na⁺ pump number is decreased, which leads to a further loss of K⁺ and gain in Na⁺ in order to contribute intracellular K⁺ to the K⁺-depleted ECF. Extracellular [Na⁺] does not change reciprocally with [K⁺] during this early phase of hypokalemia because the amount of Na⁺ in the ECF is a primary determinant of the ECF volume, a condition that keeps the concentration fairly constant.

In the first 2 days of dietary K⁺ restriction there is a more rapid decrease in ECF [K⁺] (a 20% fall) than that over the subsequent 8 days (an additional 22% fall) (Fig. 1). The reduced rate of ECF K⁺ loss beyond day 2 is not unexpected because it takes ~48 h for rats on K⁺-restricted diets to induce mechanisms to actively reabsorb K⁺ and maximally reduce urinary K⁺ excretion, mediated by the induction of renal collecting duct H⁺-K⁺-ATPase activity (26, 48).

The responses in soleus and WG are indistinguishable during the initial 2 days of K⁺ restriction (Fig. 3): both muscles lose over 36% of the initial 2-subunits, and changes in muscle [K⁺] and [Na⁺] are similar. Kjeldsen et al. (22) reported that after 3 days of K⁺ deprivation ouabain binding to whole soleus muscle (a measure of active 2-type pumps at the plasma membrane) decreased 15%. That the reduction in 2 Na⁺ pump pool sizes in soleus is greater than the decrease in surface ouabain binding sites suggests that there is some ouabain binding by 1 in soleus or that the decrease in 2 pumps may include both surface and nonsurface pools. Even though 2 levels in both muscles fall 36%, intracellular [K⁺] is only beginning to fall in soleus and does not fall significantly in WG, evidence that the decrease in 2 precedes, and likely accounts for, the decrease in muscle K⁺ stores. The finding also indicates that a loss of 36% of the muscles' Na⁺ pumps is not associated with a rapid change in cell K⁺ stores. That is, the remaining 2 and invariant 1 pumps are capable of nearly matching active K⁺ influx to passive K⁺ outflux, so that cell K⁺ stores change slowly and progressively during the 10 days of the K⁺-restricted diet. In previous studies, losses in total muscle K⁺ levels as high as 11% in whole gastrocnemius muscles after 3 days of K⁺-deficient fodder have been reported (22).

There is a provocative divergence in the responses of soleus and WG to K⁺ deprivation after 2 days (Fig. 3). In soleus, neither total 2 Na⁺ pump abundance nor cell K⁺ level decreases further between days 2-5, but both decrease between days 5 and 10. Thus changes in soleus [K⁺] mimic the changes in soleus 2 immunoreactivity, evidence for a causal link between the two. The lack of change in cell [K⁺] or [Na⁺] between days 2 and 5 of K⁺ restriction in soleus may be influenced by the fact that slow-twitch oxidative soleus muscle is more sensitive to changes in intracellular [Na⁺] than fast-twitch muscles (11), that is, the increases in muscle [Na⁺] might stimulate Na⁺ pumps and retard the loss of K⁺ more in slow-twitch oxidative soleus than in fast-twitch glycolytic WG. In WG, total 2 Na⁺ pump abundance falls dramatically throughout the 10 days of K⁺ restriction. By day 5, the fall in WG is twofold greater than that in soleus, and by day 10 only 6% of the total 2 pools remain in WG. Despite this large difference between the muscles, they both lose about the same percentage of cell K⁺. One hypothesis is that both muscles lose the same fraction of Na⁺ pumps expressed in the plasma membrane, with soleus storing pumps in endosomal pools during K⁺ restriction.

What is the magnitude of the physiological impact of the shift of K⁺ from the muscle ICF to the ECF? ECF [K⁺] would fall precipitously without the skeletal muscle adjustment because the amount of K⁺ in the ECF is small. In fact, the amount of K⁺ shifted from ICF to ECF is more than seven times the amount of K⁺ contained in the ECF of a control animal, a result calculated as follows (assumptions from Ref. 40). If we assume that ECF is 20% of the body weight, then a 280-g rat would have an ECF of 0.056 liters. Because ECF [K⁺] is 4.5 mM (Fig. 1), the control ECF contains ~0.25 mmol K⁺. Assuming that ICF is 40% of the body weight, the same rat would have an ICF of 0.112 liters. Because muscle contains ~80% of the ICF (0.090 liters) and muscle ICF [K⁺] is 120 mM (Fig. 3), muscle ICF contains 10.8 mmol of K⁺, roughly 40 times more than that in the ECF pool. After 10 days of the low-K⁺ diet, muscle loses an average of 17% of the intracellular stores (Fig. 3), equivalent to 1.84 mmol, which is more than seven times the amount contained in the ECF at day 0 (0.25 mM). In other words, the extracellular K⁺ has been replaced seven times over with K⁺ from the muscle stores after 10 days of a K⁺-restricted diet, evidence that this regulatory adjustment is critical.

The calculated ratio of 2 to 1 of near 1:1 in both muscles is not what one would predict from the relative RNA levels in control muscles. RNA ratios can be estimated directly with isoform-specific cDNA probes of similar lengths and labeled to similar ³²P specific activities. mRNA ratios of 2 to 1 in skeletal muscle have been reported to be between 2.5 (0.7 2 and 0.3 1) (13) and 4 (0.8 2 and 0.2 1) (37). However, there is no a priori reason to assume that mRNA ratios are good predictors of  protein ratios because other factors, from isoform-specific translatability to competition for heteromer formation with 1 or 2, will influence the protein ratio. Our estimate of 50% 2 in soleus agrees with what was observed by backdoor phosphorylation (15), assuming that the fraction of 2 in the euthyroid diaphragm is midway between that observed in the hypothyroid (30%) and hyperthyroid (65%) diaphragms. The percentage of 2 in soleus, calculated by Lavoie et al. (23) at between 60% in the surface membrane and 87% in intracellular membranes, is higher than our estimate. This is not unexpected because the subcellular membrane fractions assayed are expected to be enriched in 2, and it has been established that the subcellular distribution of 2 is different from that of 1 in red muscle (18). In comparison, in this study we calculated the 2-to-1 ratio in total homogenate, which contains all the cell membranes, thus averaging the ratio in membranes enriched in 2 with those enriched in 1.

Azuma et al. (5) reported that, in rats maintained on a low-K⁺ diet for 14 days, hindlimb 2 mRNA decreased 35% (1 and 1 were unchanged) and that 2 protein decreased by 82%, suggesting that changes in 2 mRNA alone could not account for the changes in 2 protein (5). Because the whole hindlimb in rats is a composite of different muscles, it was conceivable that the mRNA changes were muscle specific. We have previously determined that during the transition from euthyroid to hypothyroid states, changes in 2 mRNA levels in mixed gastrocnemius muscle predicted the changes in 2 protein levels (both decreased 45%) (6). However, unlike what was found for regulation by thyroid status, mRNA levels in either soleus, WG, or whole hindlimb measured in this study did not change during 10 days of K⁺ deprivation (Fig. 4). Thus we do not confirm the observations in our previous study (5) and hypothesize that 2 mRNA levels decrease only after a K⁺ deprivation of more prolonged duration. Alternatively, it is possible that the K⁺-deficient diet used in the present study is better matched to the control diet than in the previous report, in which the weights of the K⁺-deprived rats were significantly lower than the controls after 14 days of the low-K⁺ diet. We conclude that the decreases in Na⁺ pump expression during K⁺ deprivation can be explained by either a decrease in - and -transcript translatability and/or increased protein degradation. A rapid increase in protein degradation in muscle is not without precedent. The ubiquitin-proteasome pathway plays a role in increasing the rate of muscle protein degradation during denervation, fasting, or insulinopenia (34). Plasma membrane proteins are also degraded by internalization to endosomal pools and routing to lysosomes. The 21-type heteromers are known to shuttle between endosomal pools and the plasma membrane with insulin stimulation in oxidative muscles such as soleus but not in glycolytic muscles (24). These findings suggest the hypothesis that when ECF [K⁺] falls the rate of Na⁺ pump internalization increases in both muscle types, that a portion of the internalized pumps are stored in endosomal pools in the soleus and are available for return to the plasma membrane with K⁺ restoration or insulin stimulation, and that pumps are routed for degradation in the lysosomes in both muscles. Such a pattern would account for the smaller decrease in 2 in soleus than in WG, along with the similar rates of loss of K⁺ from both muscle types (Fig. 3).

How tissues like muscle or kidney sense the fall in extracellular [K⁺] and then effect tissue-specific changes such as the decrease in muscle Na⁺-K⁺-ATPase 2 levels remains unclear. One theory is that there are K⁺ sensors in the gut, portal circulation, and/or liver that respond to local changes in extracellular [K⁺], secondary to enteric changes (39). This suggestion complements the recent identification of the calcium-sensing receptor (CaR) (7). Indeed, Quarles et al. (38) have theorized that CaRs may represent one member of a family of "cation-sensing" cell surface receptors. Further, Hevener et al. (16) localized glucosensors to the portal vein. When these results are taken together, it is tempting to speculate that both glucose and K⁺ sensors in the hepatic portal vein or liver may respond to dietary intake, stimulating the release of humoral factors (such as insulin when glucose and K⁺ levels are elevated) that alter muscle and kidney K⁺ handling by regulating transporter levels.

In conclusion, our results demonstrate a temporal relationship between the decreases in 2 Na⁺ pump pools and a coincident or subsequent decrease in muscle [K⁺] during K⁺ deprivation in both slow-twitch oxidative and fast-twitch glycolytic muscles. The study establishes there are muscle type-specific mechanisms in place to effect the shift of K⁺ from ICF to ECF and that in both muscles the changes are independent of changes in mRNA levels.