1Department of Molecular and Cellular Physiology and 2Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Submitted 11 May 2004 ; accepted in final form 6 July 2004
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ABSTRACT |
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Na+-K+-ATPase 2 catalytic subunit; heterozygous mice; knockout mice; resting potential
The functional Na+-K+-ATPase is composed of a primary catalytic -subunit (
112 kDa) and a smaller glycosylated
-subunit (
35 kDa) that targets the
-subunit to the plasma membrane and may modulate substrate affinity (30).
-Subunits (or FxYD-domain proteins) also exist, which can interact preferentially with specific
-isozymes to regulate their catalytic properties. Multiple isoforms exist for
-,
-, and
-subunits, and numerous studies suggest that they play tissue-specific roles.
Skeletal muscles express at least two major -subunit isoforms that each show a distinct developmental and muscle type-specific expression and subcellular localization. The
1-isoform is expressed throughout muscle development, whereas the
2-isoform is expressed later and becomes the predominant subunit of adult muscle (22, 44). Human skeletal muscle also expresses the
3-isoform (41). The mouse diaphragm expresses the
1- and
2-isoforms; during development, the
2-isoform is expressed when the t tubules and dihydropyridine receptors appear (9). The
2-isoform is expressed at higher abundance in fast glycolytic compared with slow oxidative muscles, reaching up to 87 and 65% of total
content in the mouse extensor digitorum longus (EDL) and soleus (SOL), respectively (Refs. 9, 22, and 53 and Shelly, unpublished results). The
1-isoform is localized on the surface sarcolemma, and the
2-isoform is on both the surface and the t tubules at the triads in various adult muscles and species, including mice (56).
The functional roles of the -isoforms in skeletal muscle are not completely understood. Because of its ubiquitous tissue distribution, the
1-isoform is widely thought to perform a housekeeping role in maintaining equilibrium ion gradients; however, limited studies exist that address this hypothesis in skeletal muscle in the absence of
2. In addition, a dynamic role of the
1-isoform in restoring ion gradients during contraction is inferred from the 20-fold stimulation of Na+-K+ transport in contracting muscles, a rate that utilizes the total muscle Na+-K+ transport capacity (7, 13, 27). There is less consensus on proposed unique role(s) for
2-isoform in skeletal muscle. In addition to restoring ion gradients and excitability, it is proposed to modulate Ca2+ signaling and thereby contractility. The EDL and SOL of mice with genetically reduced
2-isoform content produce greater force (22), and this is thought to occur through an association of
2-containing pump units with the Na+/Ca2+ exchanger in a submembrane domain, where their combined activities can modulate dynamic Ca2+ signaling from the sarcoplasmic reticulum (SR). A similar role for
2-isoform is proposed to explain the altered contractility of heart and smooth muscles and the altered Ca2+ signaling in astrocytes of mice with genetically reduced
2-isoform content (5, 21, 26, 51). In addition to an acute cellular role, the large
2-isoform content of skeletal muscle is proposed to maintain systemic K+ homeostasis under a variety of chronic conditions of altered plasma K+ or hormonal status (24, 28, 34, 35, 54; however, see also Ref. 37).
The tissue and subcellular distribution of the 2-isoform, in particular its selective expression in electrically excitable cells (skeletal muscle, heart, nerve) or the cells that surround them (adipocytes, astrocytes), suggested to us that it could modulate excitability in skeletal muscle. In addition, the findings that the
2-isoform can modulate contractility in adult heart and skeletal muscles without change in the resting Na+ and K+ concentrations (22, 26) suggested that the
2-isoform may play a greater role in active compared with resting muscle. The activity of the Na+/Ca2+ exchanger (NCX), which is present in the t tubules (47), is low in resting muscle but becomes measurable during tetanic activity to extrude Ca2+ (4). The localization of the
2-isoform in the t tubules, where it can clear the activity-dependent K+ and Na+ and control the Na+ gradient that drives Na+/Ca2+ exchange, is optimal for modulating both excitability and contractility.
This study uses gene-targeted mice that lack one or both copies of the Na+-K+-ATPase 2-subunit gene (26) to examine the contributions of the
1- and
2-isoforms to the excitability and contractility of skeletal muscle. We tested the hypotheses that the
1-isoform alone is able to maintain the equilibrium Na+ and K+ gradients in resting muscle and that
2-isoform can modulate excitability and contractility in active muscles. We measured resting potentials, action potentials, and contractile force during tetanic and fatiguing stimulation in the diaphragm of wild-type (WT),
2-heterozygous (HET), and
2-isoform knockout (KO) mice at embryonic age (E; in days) 18.5. The
2-KO mice die just after birth, before the
2-isoform is expressed in the limb muscles; however, the perinatal diaphragm, which matures early (16) and expresses both
-isoforms in the WT, provided a physiological model for addressing these questions in a differentiated muscle (9, 39).
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METHODS |
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Electrical recording.
The diaphragm was fixed to a Sylgard (Dow Corning, Midland, MI) mount on the chamber bottom. Resting potentials and action potentials were measured from superficial fibers, diameter 1520 µm, in the central region of the muscle with the use of borosilicate glass filament microelectrodes (World Precision Instruments, Sarasota, FL). Electrodes were filled with 3 M KCl (2540 m). Membrane potentials were measured using an Axoclamp 2A amplifier (Axon Instruments, Union City, CA) in current-clamp mode. Resting potentials were read directly from the voltage output. We accepted all resting potential values that remained stable and did not depolarize on impalement. To elicit action potentials, a stimulus was delivered extracellularly with the use of a small-tip concentric bipolar electrode (FHC, Bowdoinham, ME) positioned near the muscle surface. Action potentials were captured digitally on tape and analyzed using the Axotape program (Axon Instruments). Resting potentials and single action potentials were recorded using a standard Krebs-Ringer bicarbonate buffer composed of (in mM) 154 Na, 5 K, 2 Ca, 1 Mg, 138 Cl, 25 HCO3, 1 H2PO4, and 11 glucose at 22°C. Trains of action potentials were recorded using a Cl-free buffer composed of (in mM) 166 Na, 5 K, 1 Ca, 2 Mg, 2 Cl, 78 SO4, 25 HCO3, 1 H2PO4, 11 glucose, and 54 sucrose and 50 µM dantrolene sodium at 29°C. The solutions were bubbled with 95% O2-5% CO2, pH 7.4.
Contractility measurements. Isometric contractility was measured as described previously (39). Diaphragm strips were attached to triangular clips and mounted to a force transducer in a constant-temperature sealed chamber with freshly circulating Krebs solution containing (in mM) 154.2 Na, 4.7 K, 2.5 Ca, 1.2 Mg, 127.7 Cl, 25 HCO3, 1.2 H2PO4, 1.2 SO4, 11 glucose, and 0.026 EDTA equilibrated with 95% O2-5% CO2 at 37°C. Optimal resting tension at the outset was determined empirically to be 3 mN by adjusting resting tension from zero until maximal twitch force was achieved. Diaphragm strips were field stimulated using platinum wire electrodes with capacitor discharges of equal but alternating polarity (280 Hz at 620 V, 2-ms duration). Fatigue-producing stimulation was performed by applying tetani of 1-s duration at supramaximal voltage (1618 V) and frequency (60 Hz) to the diaphragm every 0.45 s. Force was recorded using a differential capacitor force transducer (Harvard Apparatus, South Natick, MA) connected to a data-acquisition system (BioPac System, Goleta, CA). Force was normalized to the average cross-sectional area of the E18.5 diaphragm strip and expressed as a percentage of maximal tetanic force. Cross-sectional areas were measured from fixed sections using a Nikon light microscope at x100.
Western blot analysis.
Microsomal membrane samples were prepared from E18.5 diaphragms and resolved by SDS-PAGE as described previously (9). Proteins were electrophoresed for 0.31.5 h on 10% precast polyacrylamide minigels (GeneMate Express Gels) with Bio-Rad (Hercules, CA)-prestained SDS-PAGE standards, blotted onto a polyvinylidene difluoride membrane (Immobilon-P or Immobilon-PSQ for phospholamban; Millipore, Billerica, MA), and incubated overnight at 4°C with the following primary antibodies: Na+-K+-ATPase 1-isoform monoclonal at a 1:2,500 dilution (
6f; Univ. of Iowa Developmental Hybridoma Bank, Iowa City, IA); Na+-K+-ATPase
2-isoform anti-HERED polyclonal at a 1:2,500 dilution (kindly provided by Thomas Pressley, Texas Tech Univ.); Na+-K+-ATPase
3-isoform monoclonal (MA3-915; Affinity Bioreagents, Golden, CO) at a 1:1,000 dilution; sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1 monoclonal (MA3-912, Affinity Bioreagents), which recognizes splice variants 1a and 1b at a 1:5,000 dilution; SERCA2 monoclonal (MA3-919, Affinity Bioreagents), which recognizes splice variants 2a and 2b at a 1:1,400 dilution; phospholamban (PLB) monoclonal (MA3-922, Affinity Bioreagents) at a 1:5,000 dilution; NCX monoclonal (R3F1; Swant, Bellinzona, Switzerland) at a 1:500 dilution; plasma membrane Ca2+-ATPase (PMCA) monoclonal (Swant) at a 1:2,500 dilution; and calsequestrin polyclonal (PA1-913, Affinity Bioreagents) at a 1:1,000 dilution. The Na+-K+-ATPase antibodies have been shown to be isoform specific (3, 18, 55). For monoclonal primary antibodies, blots were incubated with peroxidase-conjugated goat anti-mouse secondary antibody [IgG F(ab')2; Jackson ImmunoResearch Laboratories, West Grove, PA] for 1 h at room temperature (1:40,000 dilution for Na+-K+-ATPase
1- and
3-isoform; 1:10,000 for SERCA1, SERCA2, PLB, and calsequestrin; and 1:5,000 for NCX and PMCA). For polyclonal primary antibodies, blots were incubated with anti-rabbit IgG, heavy- and light-chain goat secondary antibody (Cortex Biochem, San Leandro, CA), for 1 h at room temperature (1:40,000 dilution for Na+-K+-ATPase
2-isoform and 1:10,000 for calsequestrin). Samples were incubated at 37°C for 30 min (Na+-K+-ATPase
-isoforms, SERCA1, SERCA2, NCX, and PMCA) or boiled for 5 min (PLB). Blots were developed with the use of an enhanced chemiluminescence system (Amersham-Pharmacia, Piscataway, NJ), visualized on X-ray film (Kodak BioMax MR), and quantified by densitometry (ImageQuant; Molecular Dynamics, Sunnyvale, CA).
Data analysis.
Results are given as means ± SE; n = no. of observations. Differences between means were analyzed by one-way analysis of variance (ANOVA) for repeated measures, with statistical significance at P 0.05. Force-frequency and force-voltage data were analyzed using a two-way repeated-measures ANOVA. Western blot data were analyzed, assuming a mixed-effects model including genotype (fixed effect) and sample (random effect). The inclusion of a random sample effect allowed the estimation of two sources of variability: within samples (variance A) and between samples (variance B). The variance B of between-group means was compared with variance A for hypothesis testing. After an overall significant group effect, the equality of mean values of HET and KO compared with WT was tested using Dunnett's statistic to control for multiple comparisons against a control group.
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RESULTS |
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Diaphragm fibers of 2-HET and
2-KO mice show altered contractility.
The absence of one copy of the Na+-K+-ATPase
2-isoform results in increased twitch and tetanic force in the adult EDL muscle (22) and greater contractile force in the heart, which is associated with a larger Ca2+ change during the contraction cycle (26). Total ablation of the
2-isoform also alters agonist-induced contraction in smooth muscle at birth (51). The perinatal diaphragm of
2-KO mice generates normal twitch force in response to a single stimulus (39), but its contractility during sustained activity, the normal physiological mode of this muscle, has not been examined. Figure 5 and Table 4 show that maximum tetanic force in the
2-HET and
2-KO diaphragm is comparable with WT. In addition, the rate of contraction at half-maximum force is not altered, indicating that the speed of contractile activation is not changed. However, relaxation from contraction is significantly faster in the
2-HET and
2-KO fibers. The time to half-relaxation after a maximum tetanic contraction is significantly shorter in the
2-HET and
2-KO diaphragm and speeds relaxation by 1.7-fold (Table 4).
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Faster relaxation rate of 2-HET and
2-KO muscle does not result from compensatory changes in the expression of Ca2+ handling proteins.
Relaxation from contraction occurs when cytosolic Ca2+ is restored to resting levels. The faster relaxation of the
2-HET and
2-KO diaphragm suggests that Ca2+ removal after tetanic contraction may be enhanced when
2-isoform is absent or reduced. One mechanism by which this could occur is if the altered genotype causes compensatory changes in a protein(s) that influences Ca2+ clearance after contraction. To test this, we measured the expression of SERCA1 and SERCA2, the Ca2+ transporters of the SR, in skeletal muscle; calsequestrin, the Ca2+-binding protein of the SR lumen; PLB, a regulator of SERCA2; the PMCA; and the plasma membrane NCX.
In mammalian muscles, the rate of relaxation at early times after contraction is controlled largely by the rate of active Ca2+ transport into the SR, mediated by SERCA. After a tetanic contraction, this requires clearing cytosolic Ca2+ in the 10 µM range within 500 ms. Increased SERCA expression is associated with faster relaxation and underlies the faster relaxation of fast compared with slow fiber types (11, 57), whereas reduced SERCA expression is associated with slowed relaxation (38). The adult diaphragm of rat expresses SERCA1 and the muscle-specific SERCA2a, and these represent 75 and 23% of total SERCA expression, respectively (38). The SERCA isoform content of mouse perinatal diaphragm is not known, but other neonatal muscles express SERCA1b and SERCA2b, splice variants of SERCA1 and SERCA2. The SERCA antibodies used in this study recognize both a- and b-forms.
Figure 7 and Table 1 show that SERCA1 is expressed in the perinatal diaphragm of WT mice at levels comparable with adult. It is unchanged in the 2-HET and reduced in the
2-KO diaphragm, a direction opposite that required to speed relaxation. Because the amount of protein used to detect SERCA was small, we probed 2 µg each of the same samples with an antibody against calsequestrin as a technical control. Calsequestrin expression is comparable in all genotypes.
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PMCA is the primary extrusion pathway for smaller, sustained Ca2+ loads. Because of its low activity and high affinity (52), it is widely thought to play a greater role in resting Ca2+ homeostasis than in the rapid, dynamic Ca2+ loads during the contraction cycle. Expression of PMCA increases 1.4-fold in the 2-HET and is unchanged in the
2-KO. The small increase in the HET cannot explain a dramatically faster relaxation in both genotypes within 500 ms of a tetanus.
The plasma membrane NCX in skeletal muscle operates in forward mode during contraction and can contribute to global Ca2+ clearance (4). This contribution is small compared with SERCA because of its slow kinetics and low transport capacity (19, 23). NCX expression is low in the perinatal diaphragm of WT mice (Fig 7; note heavy loading required to detect it) and decreases by almost one-half in the 2-HET and
2-KO. A change in this direction is expected to decrease Ca2+ clearance across the plasma membrane and promote Ca2+ accumulation rather than clearance. Moreover, the resulting Ca2+ increase is not expected to change global Ca2+ due to the low NCX capacity of skeletal muscle, and this is supported by the finding that peak force is not changed under twitch or tetanic conditions.
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DISCUSSION |
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This finding was initially surprising, considering the high 2-isoform expression in WT diaphragm (9, 39). However, measurements in an independent
2-KO mouse model found no difference among the resting potentials of WT, HET, and KO diaphragm (25). In addition, the adult EDL of
2-HET mice shows no change in the resting K+ or Na+ content (22), a result that predicts no change in resting potential. To relate the present finding to that study, we measured resting potentials in adult EDL muscles of
2-HET mice. Resting potentials were 80.1 mV ± 3.8 mV (n = 20) in WT and 78.0 ± 5.5 mV (n = 20) in HET (Moseley AE, Lingrel JB, and Heiny JA, unpublished data). Thus reducing
2-isoform in adult EDL, where
2-isoform comprises up to 87% of total
-isoform content, does not significantly change the resting potential or resting ion gradients, consistent with the small effect of total
2-isoform ablation on the perinatal diaphragm. The small contribution by
2-isoform to the resting potential is statistically significant, on the basis of over 600 measurements. The absence of a contribution by
2-isoform in the study of Ikeda et al. (25) may be due to the small sample (n = 45) or to the different KO model. Together, these findings demonstrate in a physiological model that the
1-isoform alone is able to maintain equilibrium ion gradients and the resting potential of diaphragm muscle.
This conclusion does not exclude the possibility that the 1-isoform may also play a dynamic role(s) in active muscles, when the Na+ and K+ concentrations are expected to diverge from equilibrium. Indeed, our finding that a diaphragm with only
1-isoform can maintain action potentials and force under a variety of conditions demonstrates that
1-isoform makes an important contribution in active muscle. A role of both
-isoforms in active muscles is also inferred from the finding that the rate of active Na+-K+ transport increases up to 20-fold in contracting muscles (13, 27), a rate that requires utilization of up to 100% of the total
-isoform transport capacity (7).
A related conclusion is that the 2-isoform contributes significantly less to the resting potential than expected from its proportional expression. Normally, the transport activity of the pump itself, which is electrogenic, makes a direct contribution of up to 16 mV to the resting potential (42, 49). The electrogenic potential adds to the diffusion potentials for K+ and Na+ (at constant Na+ and K+ concentrations) and is a direct measure of pump activity (2, 31). Our data show that the
2-isoform contributes up to 4 mV to the resting potential of embryonic diaphragm in the
2-KO and <2 mV to the resting potential of adult EDL in the
2-HET, a muscle in which
2-isoform comprises >70% of total
-subunit. Recently Krivoi et al. (32), using a range of ouabain concentrations to selectively inhibit
1- and
2-isoforms, found that
1- and
2-containing isozymes contribute 15 and 4.5 mV, respectively, to the resting potential of adult rat diaphragm. This close agreement from independent laboratories and models argues strongly that
2-isoform contributes significantly less at rest than expected from its pump number.
2-Isoform influences spontaneous action potential activity in the perinatal diaphragm without impairing excitability to nerve input.
The lower firing frequency of spontaneously active KO fibers reveals a developmental influence of the
2-isoform on electrical activity before birth. This role is apparently minor, because it does not compromise the ability of
2-KO fibers to respond to driven input and does not impair developmental progression to any obvious degree, on the basis of multiple indexes of muscle differentiation (39). The basis of the decreased excitability during spontaneous firing is not known. It could arise from a secondary effect of
2-isoform ablation on a Na+ or K+ channel or a resting ion channel that is needed for repetitive excitation. For example, an increased conductance between action potentials could slow depolarization to threshold and limit action potential frequency.
2-Isoform modulates contractility in the diaphragm.
The major phenotypic consequence of
2-isoform ablation is a dramatically faster rate of relaxation from tetanic contraction. This result is itself surprising, since it is not common to find a selective effect on relaxation without change in contractile activation or force. All indexes of contractile activation (rate, voltage dependence) and force (twitch and tetanic) are normal in KO, except that the force-frequency relationship is shifted to higher frequencies. A shift to higher frequencies is consistent with the faster relaxation of the KO muscle. This result excludes the possibility, for example, that the change in
2-isoform may have altered contractile protein content, which is expected to alter both activation and relaxation. The dramatic and selective effect on relaxation may suggest that Ca2+ clearance across the SR or the plasma membrane is enhanced when
2-isoform is reduced or absent. However, when this question was examined carefully, we found no change in Ca2+ handling proteins that can explain the result. SERCA is the major player in clearing the large, dynamic Ca2+ change during the contraction cycle, but there is no evidence for increased expression of SERCA or PLB, its principal modulator. The expression of other Ca2+ handling proteins does not change dramatically, except for NCX expression, which is reduced by almost one-half in both HET and KO. Decreased NCX number is expected to promote Ca2+ accumulation rather than clearance. The reduction in NCX number is interesting because NCX number also changed in the heart of
2-KO mice (36) and in astrocytes of
2-HET and
2-KO mice (21), where decreased
2-isoform expression is associated with increased NCX number. The significance of these differences is not known but may suggest that control of Na+-K+-ATPase
2-isoform and NCX expression are somehow linked.
When comparing the perinatal diaphragm to other muscles with genetically altered 2-isoform expression, a common finding is that muscles with reduced or inhibited
2-isoform all show altered contractility, without change in bulk Na+ or K+ concentrations. This consistent and striking finding suggests that
2-isoform is able to influence dynamic Ca2+ signaling. It is possible that the
2-isoform may preferentially provide the Na+ gradient for Na+/Ca2+ exchange in a submembrane domain where their combined activities can modulate Ca2+ signaling. This mechanism is proposed to explain the enhanced contractility of EDL, SOL, and heart muscles of
2-HET mice (22); the altered contractility of smooth muscles of
2-KO mice (51); and the altered Ca2+ signaling in
2-KO astrocytes, in which the Na+-K+-ATPase
2-isoform and the NCX colocalize near junctions with the endoplasmic reticulum (ER) (5, 21, 26, 29, 51).
Although the contribution of NCX to rapid clearance of global Ca2+ is small compared with SERCA, the reduction in NCX number may cause Ca2+ to accumulate locally near the triads, where it can influence Ca2+ signaling from the SR (10, 33). The Na+-K+-ATPase 2-isoform and the NCX are both present in the t tubules and triads (47), and there is evidence in heart and skeletal muscle for a microenvironment near the Na+-K+-ATPase, where the local Na+ concentration differs from cytosolic levels (50). Up to 85% of the t tubule area in skeletal muscle is associated with the SR across a junctional gap of only 1214 nm (15, 17), where the local Ca2+ in the cleft may reach hundreds of micromolar (46). Thus, although the reduction in
2-isoform leads to opposite changes in NCX expression in heart and skeletal muscles, the consequence of this on the junctional Ca2+ change may be an increase in both muscles. This is because NCX can operate in reverse mode during cardiac contraction to supply trigger Ca2+, whereas in skeletal muscle it operates in forward mode to clear Ca2+ (4).
Multiple signaling and transport systems that can influence the junctional Ca2+ concentration are localized at the triad junction, including the Na+-K+-ATPase 2-subunit, the PMCA, the dihydropyridine receptor, and the ryanodine receptor (RyR). The overall consequences of reduced
2-isoform and altered NCX expression on Ca2+ signaling depend on complex interactions between the local Ca2+ change, Ca2+ release by the RyR, Ca2+ stores within the SR/ER, and Ca2+-dependent filling of the SR/ER stores, which are not well understood. It is possible that an increased junctional Ca2+ change may shorten relaxation by enhancing Ca2+-dependent inactivation of RyR1. To uncover the mechanism of the modulation of contractility in skeletal muscle by the Na+-K+-ATPase
2-isoform, an important direction for future experiments will be to determine the activity-related Ca2+ changes, both local and global, and the alterations in NCX function that occur in muscles with altered
2-isoform expression.
It is noteworthy that the inherent fatigue resistance of the perinatal diaphragm is not compromised in 2-KO fibers. This is consistent with the finding that
2-KO fibers fire repetitive action potentials without change in baseline or afterpotential (Fig. 3D) and indicates that they are able to maintain excitability despite the activity-dependent increase in extracellular K+, including K+ in the t tubule lumen. This result is puzzling because removal of
2-isoform is expected to leave the t tubules without a functional enzyme, leading to depolarization. It is possible that
1-subunit targeting is altered in the
2-KO or that the
1-subunit distributes over both surface and nonjunctional t tubule membranes. Studies of
1-isoform localization in WT muscles do not completely exclude the later possibility, given the small area of nonjunctional membrane (1015% of t tubule area in adult fast muscles). Moreover, the perinatal diaphragm, a continuously active and highly fatigue-resistant muscle, is inherently less dependent on active transport to clear dynamic K+ loads in the tubules, because the small fiber size (7- to 10-µm radius, compared with >50 µm in adult) allows for efficient clearance by diffusion alone (1). In addition, its more depolarized resting potential is protective against dynamic changes in EK that occur as extracellular K+ rises during repetitive action potential activity. The large difference between the resting potential and EK provides a greater range over which an increase in K+ or Cl permeability can hyperpolarize the membrane passively (at least up to the limit set by EK), before the concentration gradients and resting EK are fully restored by active transport.
Finally, the finding that contractility is altered in the 2-HET and
2-KO diaphragms addresses underlying questions. Why does the diaphragm express two
-isoforms? Or, in the absence of
2-isoform, can adding more
1-isoform pump units restore normal muscle function? Our results show that an increase in
1-isoform number alone, up to double that in WT diaphragm, cannot compensate fully for the lack of
2-isoform. Despite their near-normal resting potentials, excitability, and contractile activation, the
2-HET and
2-KO diaphragms do not relax normally from contraction.
Perspectives.
The distinct expression pattern and subcellular localization of the Na+-K+-ATPase -subunit isoforms in skeletal muscle suggest that they play distinct cellular roles. Results of this study and previous attempts to uncover their roles using mice with genetically altered
2-isoform content can be summarized as follows. 1) Reduction or ablation of
2-isoform, the major
-isoform of differentiated muscle, has only a minor effect on key equilibrium parameters controlled by the Na+-K+-ATPase (the resting potential and the resting Na+ and K+ gradients). This demonstrates that the Na+-K+-ATPase
1-subunit alone plays a large role in maintaining the equilibrium K+ and Na+ gradients, as widely proposed. This conclusion does not exclude additional roles for
1-isoform in active muscle. 2) The electrogenic activity of
2-isoform contributes up to 5 mV to the resting potential of skeletal muscle. This is a consistent finding in perinatal and adult skeletal muscles from two independent transgenic models and in the diaphragm of adult rats with pharmacologically inhibited
2-isoform activity. This small contribution is strikingly disproportionate to its abundance in skeletal muscles and indicates that the
2-isoform contributes less under basal conditions than expected from its pump number. 3) Skeletal muscles with reduced
2-isoform content show altered contractility (enhanced force in adult EDL or enhanced relaxation in the perinatal diaphragm). This suggests that
2-isoform can influence intracellular Ca2+ signaling. The parallel finding that contractility is altered without change in the global Na+ or K+ gradients suggests that this modulation may occur in a microdomain, as proposed for astrocytes and other muscle types. 4) The cellular consequences of genetically reduced
2-isoform content (altered contractility and/or altered excitability in development) occur in active muscles. Together with the lack of a major effect of the
2-isoform on the resting potential or resting gradients, this suggests that the
2-isoform may make a greater contribution during muscle activity at a step after membrane excitation. A challenge for future research will be to identify the factor(s) that stimulates the activity of each
-isoform during muscle contraction or other dynamic conditions of altered demand.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* T. L. Radzyukevich and A. E. Moseley contributed equally to this work.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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2. Akaike N. Contribution of an electrogenic sodium pump to membrane potential in mammalian skeletal muscle fibres. J Physiol 245: 499520, 1975.[Abstract]
3. Arystarkhova E and Sweadner KJ. Tissue-specific expression of the Na,K-ATPase 3 subunit. The presence of
3 in lung and liver addresses the problem of the missing subunit. J Biol Chem 272: 2240522408, 1997.
4. Balnave CD and Allen DG. Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse. Am J Physiol Cell Physiol 274: C940C946, 1998.
5. Blaustein MP, Juhaszova M, Golovina VA, Church PJ, and Stanley EF. Na/Ca exchanger and PMCA localization in neurons and astrocytes: functional implications. Ann NY Acad Sci 976: 356366, 2002.
6. Clausen T and Everts ME. Regulation of the Na,K-pump in skeletal muscle. Kidney Int 35: 113, 1989.[ISI][Medline]
7. Clausen T, Everts ME, and Kjeldsen K. Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle. J Physiol 388: 163181, 1987.[Abstract]
8. Coirault C, Chemla D, and Lecarpentier Y. Relaxation of diaphragm muscle. J Appl Physiol 87: 12431252, 1999.
9. Cougnon MH, Moseley AE, Radzyukevich TL, Lingrel JB, and Heiny JA. Na,K-ATPase alpha- and beta-isoform expression in developing skeletal muscles: alpha(2) correlates with t-tubule formation. Pflügers Arch 445: 123131, 2002.[CrossRef][ISI][Medline]
10. Du GG and MacLennan DH. Ca2+ inactivation sites are located in the COOH-terminal quarter of recombinant rabbit skeletal muscle Ca2+ release channels (ryanodine receptors). J Biol Chem 274: 2612026126, 1999.
11. Dulhunty AF, Banyard MR, and Medveczky CJ. Distribution of calcium ATPase in the sarcoplasmic reticulum of fast- and slow-twitch muscles determined with monoclonal antibodies. J Membr Biol 99: 7992, 1987.[ISI][Medline]
12. Ellis KO and Bryant SH. Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium. Naunyn Schmiedebergs Arch Pharmacol 274: 107109, 1972.[ISI][Medline]
13. Everts ME and Clausen T. Excitation-induced activation of the Na+-K+ pump in rat skeletal muscle. Am J Physiol Cell Physiol 266: C925C934, 1994.
14. Fournier M, Alula M, and Sieck GC. Neuromuscular transmission failure during postnatal development. Neurosci Lett 125: 3436, 1991.[CrossRef][ISI][Medline]
15. Franzini-Armstrong C. Studies of the triad. 3. Structure of the junction in fast twitch fibers. Tissue Cell 4: 469478, 1972.[ISI][Medline]
16. Franzini-Armstrong C. Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse. Dev Biol 146: 353363, 1991.[ISI][Medline]
17. Franzini-Armstrong C, Ferguson DG, and Champ C. Discrimination between fast- and slow-twitch fibres of guinea pig skeletal muscle using the relative surface density of junctional transverse tubule membrane. J Muscle Res Cell Motil 9: 403414, 1988.[ISI][Medline]
18. Gonzalez-Martinez LM, Avila J, Marti E, Lecuona E, and Martin-Vasallo P. Expression of the beta-subunit isoforms of the Na,K-ATPase in rat embryo tissues, inner ear and choroid plexus. Biol Cell 81: 215222, 1994.[ISI][Medline]
19. Gonzalez-Serratos H, Hilgemann DW, Rozycka M, Gauthier A, and Rasgado-Flores H. Na-Ca exchange studies in sarcolemmal skeletal muscle. Ann NY Acad Sci 779: 556560, 1996.[ISI][Medline]
20. Greer JJ, Allan DW, Martin-Caraballo M, and Lemke RP. An overview of phrenic nerve and diaphragm muscle development in the perinatal rat. J Appl Physiol 86: 779786, 1999.
21. Hartford AK, Messer ML, Moseley AE, Lingrel JB, and Delamere NA. Na,K-ATPase 2 inhibition alters calcium responses in optic nerve astrocytes. Glia 45: 229237, 2004.[CrossRef][ISI][Medline]
22. He S, Shelly DA, Moseley AE, James PF, James JH, Paul RJ, and Lingrel JB. The 1- and
2-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility. Am J Physiol Regul Integr Comp Physiol 281: R917R925, 2001.
23. Hilgemann DW. Unitary cardiac Na+,Ca2+ exchange current magnitudes determined from channel-like noise and charge movements of ion transport. Biophys J 71: 759768, 1996.[Abstract]
24. Hundal HS, Marette A, Mitsumoto Y, Ramlal T, Blostein R, and Klip A. Insulin induces translocation of the 2 and
1 subunits of the Na+/K+-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. J Biol Chem 267: 50405043, 1992.
25. Ikeda K, Onaka T, Yamakado M, Nakai J, Ishikawa TO, Taketo MM, and Kawakami K. Degeneration of the amygdala/piriform cortex and enhanced fear/anxiety behaviors in sodium pump 2 subunit (Atp1a2)-deficient mice. J Neurosci 23: 46674676, 2003.
26. James PF, Grupp IL, Grupp G, Woo AL, Askew GR, Croyle ML, Walsh RA, and Lingrel JB. Identification of a specific role for the Na,K-ATPase 2 isoform as a regulator of calcium in the heart. Mol Cell 3: 555563, 1999.[ISI][Medline]
27. Juel C. Potassium and sodium shifts during in vitro isometric muscle contraction, and the time course of the ion-gradient recovery. Pflügers Arch 406: 458463, 1986.[ISI][Medline]
28. Juel C, Grunnet L, Holse M, Kenworthy S, Sommer V, and Wulff T. Reversibility of exercise-induced translocation of Na+-K+ pump subunits to the plasma membrane in rat skeletal muscle. Pflügers Arch 443: 212217, 2001.[CrossRef][ISI][Medline]
29. Juhaszova M and Blaustein MP. Distinct distribution of different Na+ pump alpha subunit isoforms in plasmalemma. Physiological implications. Ann NY Acad Sci 834: 524536, 1997.[ISI][Medline]
30. Kaplan JH. Biochemistry of Na,K-ATPase. Annu Rev Biochem 71: 511535, 2002.[CrossRef][ISI][Medline]
31. Kernan RP. Membrane potential changes during sodium transport in frog sartorius muscle. Nature 193: 986987, 1962.[ISI][Medline]
32. Krivoi I, Vasiliev A, Kravtsova V, Dobretsov M, and Mandel F. Porcine kidney extract contains factor(s) that inhibit the ouabain-sensitive isoform of Na,K-ATPase (2) in rat skeletal muscle: a convenient electrophysiological assay. Ann NY Acad Sci 986: 639641, 2003.
33. Laver DR and Lamb GD. Inactivation of Ca2+ release channels (ryanodine receptors RyR1 and RyR2) with rapid steps in [Ca2+] and voltage. Biophys J 74: 23522364, 1998.
34. Marette A, Krischer J, Lavoie L, Ackerley C, Carpentier JL, and Klip A. Insulin increases the Na+-K+-ATPase 2-subunit in the surface of rat skeletal muscle: morphological evidence. Am J Physiol Cell Physiol 265: C1716C1722, 1993.
35. McDonough AA, Thompson CB, and Youn JH. Skeletal muscle regulates extracellular potassium. Am J Physiol Renal Physiol 282: F967F974, 2002.
36. McDonough AA, Velotta JB, Schwinger RH, Philipson KD, and Farley RA. The cardiac sodium pump: structure and function. Basic Res Cardiol 97, Suppl 1: I19I24, 2002.[Medline]
37. McKenna MJ, Gissel H, and Clausen T. Effects of electrical stimulation and insulin on Na+-K+-ATPase ([3H]ouabain binding) in rat skeletal muscle. J Physiol 547: 567580, 2003.
38. Mitchell-Felton H, Hunter RB, Stevenson EJ, and Kandarian SC. Identification of weight-bearing-responsive elements in the skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene. J Biol Chem 275: 2300523011, 2000.
39. Moseley AE, Lieske SP, Wetzel RK, James PF, He S, Shelly DA, Paul RJ, Boivin GP, Witte DP, Ramirez JM, Sweadner KJ, and Lingrel JB. The Na,K-ATPase 2 isoform is expressed in neurons, and its absence disrupts neuronal activity in newborn mice. J Biol Chem 278: 53175324, 2003.
40. Muniak CG, Kriebel ME, and Carlson CG. Changes in MEPP and EPP amplitude distributions in the mouse diaphragm during synapse formation and degeneration. Brain Res 281: 123138, 1982.[Medline]
41. Murphy KT, Snow RJ, Petersen AC, Murphy RM, Mollica J, Lee JS, Garnham AP, Aughey RJ, Leppik JA, Medved I, Cameron-Smith D, and McKenna MJ. Intense exercise up-regulates Na+,K+-ATPase isoform mRNA, but not protein expression in human skeletal muscle. J Physiol 556: 507519, 2004.
42. Nikolsky EE, Zemkova H, Voronin VA, and Vyskocil F. Role of non-quantal acetylcholine release in surplus polarization of mouse diaphragm fibres at the endplate zone. J Physiol 477: 497502, 1994.[Abstract]
43. Oba T. Influence of temperature and external Ca2+ concentration upon dantrolene action on excitation-contraction coupling in frog skeletal muscle. Can J Physiol Pharmacol 59: 358363, 1981.[ISI][Medline]
44. Orlowski J and Lingrel JB. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha isoform and beta subunit mRNAs. J Biol Chem 263: 1043610442, 1988.
45. Overgaard K, Nielsen OB, Flatman JA, and Clausen T. Relations between excitability and contractility in rat soleus muscle: role of the Na+-K+ pump and Na+/K+ gradients. J Physiol 518: 215225, 1999.
46. Rios E and Stern MD. Calcium in close quarters: microdomain feedback in excitation-contraction coupling and other cell biological phenomena. Annu Rev Biophys Biomol Struct 26: 4782, 1997.[CrossRef][ISI][Medline]
47. Sacchetto R, Margreth A, Pelosi M, and Carafoli E. Colocalization of the dihydropyridine receptor, the plasma-membrane calcium ATPase isoform 1 and the sodium/calcium exchanger to the junctional-membrane domain of transverse tubules of rabbit skeletal muscle. Eur J Biochem 237: 483488, 1996.[Abstract]
48. Sejersted OM and Sjogaard G. Dynamics and consequences of potassium shifts in skeletal muscle and heart during exercise. Physiol Rev 80: 14111481, 2000.
49. Sellin LC and Sperelakis N. Decreased potassium permeability in dystrophic mouse skeletal muscle. Exp Neurol 62: 605617, 1978.[CrossRef][ISI][Medline]
50. Semb SO and Sejersted OM. Fuzzy space and control of Na+,K+-pump rate in heart and skeletal muscle. Acta Physiol Scand 156: 213225, 1996.[CrossRef][ISI][Medline]
51. Shelly DA, He S, Moseley A, Weber C, Stegemeyer M, Lynch RM, Lingrel J, and Paul RJ. Na+ pump 2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice. Am J Physiol Cell Physiol 286: C813C820, 2004.
52. Strehler EE and Zacharias DA. Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev 81: 2150, 2001.
53. Thompson CB, Dorup I, Ahn J, Leong PK, and McDonough AA. Glucocorticoids increase sodium pump 2- and
1-subunit abundance and mRNA in rat skeletal muscle. Am J Physiol Cell Physiol 280: C509C516, 2001.
54. Tsakiridis T, Wong PP, Liu Z, Rodgers CD, Vranic M, and Klip A. Exercise increases the plasma membrane content of the Na+-K+ pump and its mRNA in rat skeletal muscles. J Appl Physiol 80: 699705, 1996.
55. Urayama O, Shutt H, and Sweadner KJ. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. J Biol Chem 264: 82718280, 1989.
56. Williams MW, Resneck WG, Kaysser T, Ursitti JA, Birkenmeier CS, Barker JE, and Bloch RJ. Na,K-ATPase in skeletal muscle: two populations of beta-spectrin control localization in the sarcolemma but not partitioning between the sarcolemma and the transverse tubules. J Cell Sci 114: 751762, 2001.
57. Zubrzycka-Gaarn E, Korczak B, Osinska H, and Sarzala MG. Studies on sarcoplasmic reticulum from slow-twitch muscle. J Muscle Res Cell Motil 3: 191212, 1982.[ISI][Medline]