Contribution of Elevated Peripheral Resistance to Acute Volume Loaded Hypertension in Dogs

ABSTRACT

The role of peripheral resistance in volume loaded hypertension was examined by measuring changes in hemodynamic and humoral factors during dextran infusion for one hour, and for three hours after stopping the infusion in 30 anesthetized dogs. The mean blood pressure was elevated significantly (118%, p<0. 01), accompanied by increased cardiac output (170%, p<0. 01) during volume loading. In the recovery period, cardiac output returned to the basal level while the total peripheral resistance gradually increased (118%, p<0. 01)contributing to the maintenance of high blood pressure (108%, p<0. 01). There was no significant correlation between the hematocrit change and the increase in peripheral resistance in the recovery period. The levels of plasma norepinephrine, epinephrine, renin activity and vasopressin were suppressed by volume loading. These results demonstrated the importance of increase in peripheral resistance for maintaining high blood pressure in this acute volume excess model. The participation of the renin-angiotensin system, the sympathetic nervous system and vasopressin were all excluded and vascular structural changes were most unlikely to be involved in this acute experiment. It remains unknown whether this conversion is attributable to the summation of local autoregulation or to some other factors.     

Ouabain-Like and Non-Ouabain-Like Factors in Plasma of Patients with Essential Hypertension

Circulating inhibitor of Na⁺, K⁺-ATPase and ouabain-like immuno-reactivity were studied in patients with essential hypertension. In the plasma of patients, two types of Na⁺, K⁺-ATPase inhibitors (ouabain-like and non-ouabain-like inhibitors) and ouabain-like immunoreactivity were detected. Ouabain-like inhibitor was clearly detected at a low KC1 concentration (0.1 mM) in the assay buffer, and non-ouabain-like inhibitor was detected at a high KC1 concentration (10 mM). The plasma level of ouabain-like inhibitor correlated significantly with that of ouabain-like immunoreactivity (p<0.001) and with a mean blood pressure (p<0.01). The plasma level of non-ouabain-like inhibitor was not correlated with the levels of either ouabain-like immunoreactivity or mean blood pressure. The level of plasma ouabain-like inhibitor did not correlate with that of plasma non-ouabain like inhibitor. Both ouabain-like inhibitor and ouabain-like immunoreactivity in the plasma of patients with essential hypertension were significantly higher than those in normotensive subjects, but the plasma level of non-ouabain-like inhibitor in patients with essential hypertension was not higher than that in normotensive subjects. These results suggest that the plasma from patients with essential hypertension contains ouabain-like factor(s) which is important to mantain the high blood pressure.     

In Vitro Action of Insulin on Erythrocyte Sodium Transport Mechanisms: Its Possible Role in the Pathogenesis of Arterial Hypertension

The effects of insulin on sodium and potassium metabolism have been well known for many years; clinical observation and laboratory experience showed different results about the insulin effect on the sodium-potassium pump. Moreover, studies about the insulin effect on the sodium-potassium cotransport are not available. Therefore, the effects of insulin on Na,⁺K⁺ pump and Na⁺,K⁺ cotransport were evaluated. Results show that insulin inhibits Na⁺, K⁺ pump, while Na⁺, K⁺ cotransport is markedly activated. The possible link between pathogenesis of arterial hypertension in hyperinsulinemic subjects and present data is examined.     

Assay of a Circulating Sodium Pump Inhibitor in Patients with Essential Hypertension and Normotensive Subjects

The plasma levels of a sodium pump inhibitor (Na⁺PI) were measured by a modified method of Hamlyn et al, using dog kidney Na⁺, K⁺-ATPase. When the level of Na⁺PI was expressed as the % inhibition of the enzyme and compared with that of a control solution, it was found to be 9.0 ± 0.7% in 43 untreated patients with essential hypertension. This was significantly higher than 5.0 ± 0.4% for 56 normotensive subjects (p < 0.01). Male patients with essential hypertension showed the highest mean value of 10.5 ± 1.1%, disclosing an apparent sex difference in the patient group (p< 0.01). Only in female patients was there a significant positive correlation between the inhibitor's level and the mean blood pressure (r = 0.649, p < 0.01). These results provided additional evidence for increased Na⁺ PI in the plasma of patients with essential hypertension, which might bear an important role in the pathogenesis of the disease.     

Evidence For A Circulating Sodium-Potassium Pump Inhibitor in Low Renin Hypertension

In 1978, we reported that supernate of boiled plasma from acutely volume expanded dogs and rats reduces sodium-potassium pump activity when applied to the tail artery from a normal rat and then in 1980 we reported that the same is the case for plasma supernate from the dog with one-kidney, one wrapped hypertension, a model of low renin hypertension. Since then, we and a number of other investigators have described sodium-potassium pump inhibitory activity in the plasma of animals and humans with hypertension, particularly of the low renin variety. The activity results from a heat stable small molecule, probably the putative natriuretic hormone. It appears to be released from the hypothalamus in response to cardiopulmonary vascular distention subsequent to failure of the kidney to excrete the prevailing sodium and water intake. It probably acts on blood vessels both directly (electrogenic depolarization) and indirectly (inhibition of norepinephrine reuptake into adrenergic nerve terminals). While it may be sufficient by itself to raise blood pressure, it may be most effective when superimposed on vascular smooth muscle cells which are abnormally permeable to sodium. Efforts to determine its chemical structure should be intensified.     

Increased Dietary Potassium and Magnesium Attenuate Experimental Volume Dependent Hypertension Possibly Through Endogenous Sodium‐Potassium Pump Inhibitor

We and others have shown that inhibition of cardiovascular muscle (CVM) cell Na⁺,K‐ATPase activity (NKPTA) due to increased level of endogenous sodium potassium pump inhibitor (SPI) is involved in the mechanism of volume expanded (VE) experimental and human essential hypertension (HT). Since diets fortified with very high potassium (K) or very high magnesium (Mg) decrease blood pressure (BP), we have examined the effect of a moderate increase in dietary K alone and a moderate increase in dietary K and Mg on plasma levels of SPI, CVM cell NKPTA, and BP in reduced renal mass (RRM)‐salt HT rats, a classical model of VE HT. Seventy Percent‐RRM rats were divided in four dietary groups, (1) Na free and normal K and Mg (0Na–K–Mg); (2) normal Na, K and Mg (Na–K–Mg); (3) normal Na and high K (2 ×normal), and normal Mg (Na–2K–Mg); and (4) normal Na and high K (2 ×normal), and high Mg (2 ×normal) (Na–2K–2Mg). As expected, compared to control 0Na–K–Mg rats, Na–K–Mg rats developed HT. Blood pressure increased significantly less in Na–2K–Mg rats whereas, BP did not increase in Na–2K–2Mg rats. Hypertension in NA–K–Mg rats was associated with an increase in plasma SPI and digitalis like factor (DIF) and a decrease in renal and myocardial NKPTA. However, doubling the Mg along with K in the diet (Na–2K–2Mg) normalized SPI and DIF and increased myocardial and renal NKPTA, compared to control 0Na–K–Mg rats. Also, compared to 0Na–K–Mg rats, water consumption, urine excretion, urinary sodium excretion urinary potassium excretion (U_NaV), and (U_KV) increased in the other three groups, more so in Na–2K–2Mg rats. These data show that K and Mg have additive effects in preventing an increase in SPI, thus probably preventing the BP increase in RRM rats.     

Circulating Digitalis-Like Compounds in Essential Hypertension

Inhibitors of the Na⁺ pump have been proposed as participating in sodium excretion, xtracellular fluid regulation, and in the rise of blood pressure.

The presence of digitalis-like compounds in human plasma has been investigated by comparing the effects of plasma extracts to those of ouabain in 4 tests.

-competition with ouabain for binding to the Na⁺ pump

-inhibition of Na⁺ and K⁺ dependent hydrolysis

-inhibition of serotonin uptake by human platelets

-central hypertensive effect

Plasma fractions exhibited digitalis-like properties in the 4 tests. The effects of plasma extracts of 42 normotensive subjects (21 with family history of hypertension) and 38 patients with essential hypertension (15 with antihypertensive treatment) and 9 patients with chronic renal failure were compared.

Plasma from Forty per cent of untreated hypertensive patients and normotensives with hypertensive heredity had a high inhibition level. Inhibition was enhanced in beta-blocker treated patients and decreased in those on diuretics.

No digitalis-like activity was observed in uremic plasma.

These observations strongly suggest the presence of digitalis-like compound(s) in human plasma and point to its possible association with hypertension.     

Erythrocyte Sodium - Lithium Countertransport in Chinese: Its Relationship to Family History of Hypertension

Rates of sodium (Na⁺) - stimulated lithium (Li ⁺) efflux (Na⁺-Li⁺ countertransport) and ouabain-sensitive Na⁺ efflux (Na⁺ pump) were determined in erythrocytes of Chinese normotensive and hypertensive subjects. Near-maximal rate of Na⁺ - Li⁺ countertransport was found to be significantly higher in hypertensive than normotensive subjects. No significant difference was observed for the rate of Na⁺ pump between them. A second series of study involved normotensive subjects without and with hypertensive parent(s) (group A and B, respectively) and hypertensive subjects (group C). We found that the rate of Na⁺ - Li⁺ countertransport in group A was significantly lower than that of group B and C, while no difference existed between group B and C. No significant difference was observed for the rate of Na⁺ pump among the three groups. Our results suggested that Na⁺-Li⁺ countertransport activity could be a genetic marker for essential hypertension in Chinese, similar to that as proposed in Caucasians.     

Mechanism of hCG-induced spermiation in the toad Rhinella arenarum (Amphibia, Anura)

In Rhinella arenarum spermiation occurs as a consequence of LH/FSH increase during the amplexus or by a single dose of hCG, among other gonadotropins. The present study employs an in vitro system to study the mechanism of action of hCG in the spermiation of R. arenarum. Testicular fragments were incubated for 2 h at 28 °C in the presence or absence of 20 IU hCG with or without different PKA/PKC inhibitors and activators as well as ouabain and amiloride as Na⁺/K⁺ ATPase and transcellular Na⁺ transport inhibitors, respectively. Ouabain did not induce spermiation in absence of hCG and inhibited hCG-induced spermiation in a dose-dependent manner, reaching 90% inhibition with the higher concentration. In contrast, amiloride neither affected spermiation nor steroidogenesis. Activation of PKA with 8Br-cAMP induced spermiation in the absence of hCG while its inhibition with H89 blocked hCG action. On the other hand, PKC inhibition with Bi or STP did not affect hCG-induced spermiation although PKC activation significantly decreased hCG-dependent sperm release. These results suggest that PKC inhibits spermiation but also that the inhibition exerted by the kinase could be blocked by hCG. Taken together, these observations could indicate that PKA is involved in the mechanism of the gonadotropin action, mechanism also requiring the activation of a non-pumping Na⁺/K⁺ ATPase pathway.     

Atrial Natriuretic Factor Release During Volume Expansion in the Spontaneously Hypertensive Rat - Effect of Long-Term Hydralazine Treatment

We tested the ability of spontaneously hypertensive rats (SHR) to release atrial natriuretic factor (ANF) during acute volume loading and its relationship to right and left atrial pressures. The effect of decreasing cardiac afterload by hypotensive treatment was also investigated. Fourteen to 15-week-old SHR and Wistar-Kyoto (WKY) rats were treated with hydralazine (12 mg/kg.day p.o.) for a period of 4 weeks. Untreated rats served as controls. The systolic blood pressure (BP) of SHR decreased to normotensive levels and cardiac hypertrophy was also reduced. Isotonic, iso-oncotic volume expansion (VE) was performed 3 times as 10% increments and at 15-min intervals. Despite greater changes in left-ventricular end-diastolic pressures (LVEDP) and to a lesser extent in central venous pressure (CVP) in SHR controls, the increases in plasma ANF (N-terminal concentrations) induced by VE were of a similar magnitude in both SHR and WKY control rats. The LVEDP and ANF C-terminal elevations were of a lower magnitude in treated SHR. Auricular ANF concentrations, measured at the end of VE, were lower in the left and higher in the right atrium in SHR versus WKY. It is concluded that despite a lower distensibility of their left atrium, ANF release is not impaired in SHR upon a VE. This adequate ANF release is obtained through higher increases in atrial pressures.     

The Effect of High Salt Intake on Na+, K+-Atpase Activity of Tissues in the Rat

1. The effect of chronic feeding of high salt diet on Na⁺, K⁺-ATPase activity of heart, liver, skeletal muscle, kidney and aorta was studied in the rat.

2. Groups of rats were either given tap water or 18 g/L saline to drink. After 7 days, 3 months or 12 months, the control group and salt loaded     

Decreased Na+ K +ATPase Activity in the Aortic Endothelium and Smooth Muscle of the Spontaneously Hypertensive Rats

The activity of Na⁺ K⁺ ATPase in the endothelium and smooth muscle of the aortae of normotensive and hypertensive rats was investigated. The enzyme activity in the endothelium and smooth muscle of the spontaneously hypertensive rats (SHR) was 2.15 ± 0.48 and 12.98±0.99 respectively. These values were significantly lower (P<0.05) than the enzyme activity in the corresponding tissues (10.10 ± 1.78 for endothelium, 20.77 ± 2.54 for smooth muscle) of the normotensive Wistar Kyoto (WKY) rats. However, with the low blood pressure spontaneously hypertensive rats (LBP-SHR) i.e. in those animals whose blood pressures were below 150 mm Hg, the enzyme activity in both tissues was not significantly different from those of the WKY. Since Na⁺ K⁺ ATPase is coupled to the sodium-potassium pump whose activity affects the functions of other pumps, the results indicate that the development of high blood pressure in the SHR may be related to an alteration in the transport of cations across the cell membrane.     

A Non-Ouabain Na/K Atpase Inhibitor Isolated from Bovine Hypothalamus. Its Relation to Hypothalamic Ouabain

We have isolated from bovine hypothalamic and pituitary tissue the sodium pump inhibitor HHIF that is structurally different from ouabain. By mass spectrometric analysis this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from ouabain. It has been previously shown that HHIF inhibits the Ca²⁺-ATPase of the plasma membrane of synaptosomes. HHIF increases free calcium levels in cultured rat mesangial cells as well as mesangial cell contraction and proliferation. With the same purification procedure we have isolated in parallel HHIF and Ouabain from central nervous tissue. Ouabain elutes prior to HHIF in the final purification HPLC systems. This endogenous Ouabain has, in all the systems tested, the same chromatographic behavior that synthetic cold or [³H] Ouabain     

Insulin down-regulates the Na/K ATPase in enterocytes but increases intestinal glucose absorption

The effect of insulin on [¹⁴C] 3-O-methyl-d-glucose (3OMG) absorption in the rat jejunum was studied using an in situ perfusion technique. Insulin increased apical glucose entry into the cells and decreased intestinal retention suggesting that serosal glucose transport was enhanced by the hormone. This enhanced uptake was ascribed to an increase in the expression of glucose transporters as confirmed by Western blot analysis and not to a higher sodium gradient, since insulin reduced the activity and protein expression of the Na⁺/K⁺ ATPase. To separate the glycemic from the insulinemic effect on glucose transport, the effect of the hormone was investigated in vitro using cultured Caco-2 cells. The cells also showed an increase in [¹⁴C] 3OMG uptake and intracellular glucose levels when treated with insulin and a lower Na⁺/K⁺ ATPase activity. Phloretin, an inhibitor of GLUT2 was used to determine if these transporters are targeted by the hormone. The results showed that the effect of insulin on glucose uptake and intracellular glucose was still enhanced in presence of phloretin. Considering the inhibitory effect of the hormone on the Na⁺/K⁺ ATPase, it was concluded that insulin acts by increasing the number of glucose transporters, a hypothesis that was confirmed by Western blot analysis.     

Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart

Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na⁺/K⁺-ATPase activity and the number of receptors decreased in isolated cardiomyoctes of diabetic rats 8 weeks after streptozotocin administration. The Ca²⁺ uptake of both sarcoplasmic reticulum and mitochondria decreased simultancously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca²⁺ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.     

Na/K-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes

The positive inotropic effect produced by Na⁺/K⁺-ATPase inhibition has been used for the treatment of heart failure for over 200 years. Recently, administration of toxic doses of ouabain has been shown to induce cardiac myocyte apoptosis. However, whether prolonged administration of non-toxic doses of ouabain can also promote cardiac myocyte cell death has never been explored. The aim of this study was to assess whether non-toxic doses of ouabain can induce myocyte apoptosis and if so, to examine the underlying mechanisms. For this purpose, cardiac myocytes from rat and cat, two species with different sensitivity to digitalis, were cultured for 24 h in the presence or absence of 2 µM (rat) and 25 nm-2 µM ouabain (cat). Cell viability and apoptosis assays showed that ouabain produced, in the rat, a 43 ± 5% decrease in cell viability due to apoptosis (enhanced caspase-3 activity, increased Bax/Bcl-2 and TUNEL-positive nuclei) and necrosis (LDH release and trypan blue staining). Similar results were obtained with 25 nM ouabain in the cat. Ouabain-induced reduction in cell viability was prevented by the NCX inhibitor KB-R7943 and by the CaMKII inhibitors, KN93 and AIP. Furthermore, CaMKII overexpression exacerbated ouabain-induced cell mortality which in contrast was reduced in transgenic mice with chronic CaMKII inhibition. However, KN93 failed to affect ouabain-induced inotropy. In addition, whereas ERK½ inhibition with PD-98059 had no effect on cell mortality, PI3K inhibition with wortmannin, exacerbated myocyte death. We conclude that ouabain triggers an apoptotic cascade that involves NCX and CaMKII as a downstream effector. Ouabain simultaneously activates an antiapoptotic cascade involving PI3K/AKT which is however, insufficient to completely repress apoptosis. The finding that KN93 prevents ouabain-induced apoptosis without affecting inotropy suggests the potential use of CaMKII inhibitors as an adjunct to digitalis treatment for cardiovascular disease.     

Erythrocyte Membrane Abnormalities in Hypertension: A Comparison Between two Animal Models

Calcium-membrane interactions have been studied in two animal models of hypertension using the erythrocyte membrane as a model system. The Okamoto-Aoki strain of spontaneously hypertensive rats (SHR) was the first examined, and the activities of the Ca⁺⁺/Mg⁺⁺-ATPases in the membrane of SHR erythrocytes were found to be consistently higher than those of the normotensive controls (WKY), while other membrane enzymes such as Na⁺/K⁺-ATPase and acetylcholinesterase were not detectably altered. Erythrocyte membranes of the SHR also have a higher passive permeability to calcium as well as other functional and compositional differences when compared to those of the WKY. These findings suggest that the membrane alterations in the SHR may be related to an increased passive permeability to calcium, and a possibly compensatory increase in Ca⁺⁺/Mg⁺⁺-ATPase activity in these animals. The second animal model examined was the deoxycorticosterone/salt-induced hypertension (DOCA) in uni-nephrectomized rats. In DOCA rats with comparable degree of blood pressure elevation, none of the erythrocyte membrane abnormalities observed in the SHR were present, suggesting that the latter alterations are probably genetically determined and not a consequence of elevated arterial pressure.     

Apparent Upregulation of Na+, K+ Pump Sites in SHR Skeletal Muscle with Reduced Transport Capacity

Slow-twitch, oxidative skeletal muscles in SHR exhibit several physiological defects, including a reduced ability to maintain force during high frequency repetitive stimulation (1). Muscle fatigue may be produced by one of a variety of factors acting at different levels of the neuromuscular system. Several lines of evidence, however, suggest that SHR soleus fatigues more rapidly than WKY soleus because SHR muscles allow more K⁺ to accumulate in the extracellular space during repetitive muscle activity. An increase in extracellular K⁺ can lead to a failure in the generation or conduction of muscle action potentials. Comparison of the compound action potentials recorded from SHR and WKY muscles during repetitive stimulation provided evidence for a decrease in excitability of SHR soleus. Since the K⁺ released from muscle fibers during exercise is returned to the fiber principally via the activity of the Na⁺, K⁺ pump, the increase in extracellular K⁺ in SHR muscle may reflect a decrease in pump capacity. Measurements including intracellular K⁺ and Na⁺ content at rest, the level of hyperpolarization produced by the addition of epinephrine and insulin to SHR soleus and the post-exercise recovery of resting membrane potentials all appear to indicate that Na⁺, K⁺ pump capacity is reduced in SHR soleus muscles. Nonetheless, ouabain binding studies show a significantly greater number of pump sites in SHR muscles. The data suggest that Na⁺ pump activity is decreased in SHR soleus muscles without an apparent reduction in either the number of pump sites or in pump binding affinity.     

Prolactin increases Na+ transport across adult bullfrog skin via stimulation of both ENaC and Na+/K+-pump

PRL is involved in osmoregulation in lower vertebrates. Its serum concentration starts to increase during the metamorphosis of bullfrog tadpoles. Adult bullfrog skin transports Na(+) from the apical to the basolateral side across the skin. PRL is involved in the regulation of this transport. We investigated the effect of ovine PRL on the epithelial Na(+) channel (ENaC), Na(+)/K(+)-pump, and basolateral K(+) channels, which regulate Na(+) transport across adult bullfrog skin, by measuring the short-circuit current (SCC). At 0.1 microg/ml, PRL had no effect on the SCC. PRL (1 microg/ml) was sufficient to stimulate the SCC since 1 and 10 microg/ml of PRL each increased SCC 1.8-fold. Current-fluctuation analysis revealed that PRL (10 microg/ml) increased the density of active ENaC almost 1.8-fold. The effect of PRL on the Na(+)/K(+)-pump was investigated using apically nystatin-permeabilized skin with Ca-free Na-Ringers' solution on each side. PRL (10 microg/ml) increased SCC in this condition around 1.1-fold, suggesting that PRL stimulates the Na(+)/K(+)-pump [although PRL (1 microg/ml) had no effect on this SCC]. The effect of PRL on basolateral K(+) channels was investigated using apically nystatin-permeabilized skin with high-K Ringer's solution on the apical side. PRL (10 microg/ml) had no effect on the SCC, suggesting that PRL does not affect basolateral K(+) channels. Thus, although PRL stimulates the Na(+)/K(+)-pump, this effect probably contributes less than that on ENaC to the regulation of Na(+) transport across adult bullfrog skin.     

Displacement of the Na+/K+ pump’s transmembrane domains demonstrates conserved conformational changes in P-type 2 ATPases

Cellular survival requires the ion gradients built by the Na⁺/K⁺ pump, an ATPase that alternates between two major conformations (E1 and E2). Here we use state-specific engineered-disulfide cross-linking to demonstrate that transmembrane segment 2 (M2) of the pump’s α-subunit moves in directions that are inconsistent with distances observed in existing crystal structures of the Na⁺/K⁺ pump in E1 and E2. We characterize this movement with voltage-clamp fluorometry in single-cysteine mutants. Most mutants in the M1–M2 loop produced state-dependent fluorescence changes upon labeling with tetramethylrhodamine-6-maleimide (TMRM), which were due to quenching by multiple endogenous tryptophans. To avoid complications arising from multiple potential quenchers, we analyzed quenching of TMRM conjugated to R977C (in the static M9–M10 loop) by tryptophans introduced, one at a time, in M1–M2. This approach showed that tryptophans introduced in M2 quench TMRM only in E2, with D126W and L130W on the same helix producing the largest fluorescence changes. These observations indicate that M2 moves outward as Na⁺ is deoccluded from the E1 conformation, a mechanism consistent with cross-linking results and with proposals for other P-type 2 ATPases.

P-type ATPases are enzymes that catalyze formation of ion or lipid gradients across membranes in all phyla. These membrane proteins use homologous E1–E2 mechanisms (Fig. 1A) in which uphill transport of a subtype-specific substrate is powered by ATP hydrolysis (1). In nearly all animal cells the Na⁺/K⁺ pump, a P-type 2C ATPase, exports three Na⁺ and imports two K⁺ into the cell, against their electrochemical gradients, thereby building the ion gradients that energize other essential membrane processes, including electric signaling and secondary-active transport. This ion pump is formed by a catalytic α-subunit, homologous to the catalytic subunits of all other P-type ATPases, and an auxiliary β-subunit. The α-subunit contains the ion-transport sites within its 10 transmembrane α-helices (M1–M10) and the nucleotide (N), phosphorylation (P), and actuator (A) domains formed by its intracellular loops, while the β-subunit, with a single transmembrane segment and a large extracellular globular domain, is necessary for ion occlusion (2) and membrane stability (3). In each catalytic cycle, the pump transits through a set of partial reactions while occupying two major conformations E1 and E2, in what is also known as the Post-Albers mechanism (Fig. 1A).Open in a separate windowFig. 1.(A) Post-Albers kinetic scheme of the Na⁺/K⁺ pump (clockwise forward direction). The pump alternates between two major conformations, E1 and E2, which can be phosphorylated (P) or dephosphorylated. Parentheses indicate ions occluded within the protein. Transitions within the red box E1P(3Na⁺) ↔ E2P + 3 Na⁺_o produce transient charge movement. (B and C) Overall change in structure between Na⁺/K⁺ pump structures E1(3Na⁺) (PDB ID 3WGV; cyan) and E2P-ouabain (PDB ID 4XE5; pink) shown from two angles after aligning through the C-terminal end of the Na⁺/K⁺ pump α-subunit, (B) A lateral view, approximately parallel to the membrane plane (indicated by gray dotted lines), and (C) a view from the extracellular side, approximately perpendicular to the membrane (rotated 90° from the view in B). Residues and distances addressed in the discussion are labeled.Utilizing a combination of generic P-type ATPase as well as pump-specific inhibitors, all intermediate states of the sarcoplasmic/endoplasmic reticulum ATPase (SERCA, another P-type 2 ATPase) have been determined by X-ray crystallography (4–14), but only a handful of states have been resolved for the Na⁺/K⁺ pump (15–20). Considering the high sequence similarity and conserved catalytic cycle, it was expected that the main conformational changes should also be conserved. However, comparison of the available crystal structures in equivalent states reveals significant differences, particularly in the movement of the M1–M2 stalk. In SERCA, transitions between E1P(2Ca²⁺) and E2P states involve kinking as well as movement of the luminal part of M1–M2 perpendicular to the membrane plane (9), a movement that seems largely absent in the available Na⁺/K⁺ pump crystal structures (20) (SI Appendix, Fig. S1). Fig. 1B compares two states of such Na/K pump structures: the aluminum fluoride-inhibited E1 with 3 Na⁺ bound (ref. 20; cyan carbons) and the ouabain- and Mg²⁺-bound beryllium fluoride-inhibited structure (ref. 21; pink carbons). The closest physiologic states to these inhibitor-locked structures are enclosed with a dotted box in the catalytic cycle (Fig. 1A). Displacements in the membrane plane are absent in the side view (Fig. 1B and SI Appendix, Fig. S1), despite large movements in other directions, which are obvious in the external view perpendicular to the membrane (Fig. 1C).The dislodgment of M1–M2 is thought to guide opening of the SERCA ion-binding sites as Ca²⁺ is deoccluded and released toward the luminal side prior to occlusion of the countertransported H⁺. Because a similar mechanism has been proposed for the gastric H⁺/K⁺ pump (22), the absence of such conserved conformational change in the closely related Na⁺/K⁺ pump is perplexing, and this movement has never been evaluated in a functional Na⁺/K⁺ pump in the membrane environment. Here we address the movement of the M1–M2 region in functional Na⁺/K⁺ pumps utilizing cross-linking of engineered disulfides under voltage clamp, as well as voltage-clamp fluorometry (VCF), under conditions in which we can control the occupancy of phosphorylated states in the pump cycle. Our results indicate that the movement of the externalmost part of the α-subunit’s M1–M2 is required for Na⁺/K⁺ pump to function and, thus, conserved through the P-type 2 ATPase family. Furthermore, we illustrate a slight modification to the usual VCF approach, where TMRM is attached to a static residue while relative distance changes are evaluated by scanning with quenching tryptophans in the moving region, allowing one to study both the movement and the structural characteristics of transmembrane segments and their loops in a functional Na⁺/K⁺ pump, a powerful modification that can be exploited to investigate other transport proteins as well.