Development of (Na+ -K+)-ATPase in rat hindbrain: increments in parallel with Na+-dependent phosphorylation and K+-pnitrophenylphosphatase.

Rat hindbrain NaI-enriched microsomal (Na⁺-K⁺)-ATPase activity, K⁺-pNPPase activity, and Na⁺-dependent steady-state phosphorylation levels all increase approx 10-fold relative to microsomal protein between 5 days prenatally and 60 days postnatally. These activities, as well as the mean wet weight of the hindbrain, are at half of their 60 day values shortly after the 10th postnatal day. For all ages, these hindbrain activities average over twice those found in the forebrain in a companion paper (Bertoni & Siegel , 1978). Increases during development in the amount of the related phosphorylatable polypeptide, estimated by densitometry of stained polyacrylamide gels containing fixed amounts of microsomal protein dissolved in SDS, are in agreement with increases in steady state levels of Na⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na⁺-dependent rises steadily during development consistent with, but not obligatorily due to, a conversion of some of the previously Na⁺-independent portion. Mg2⁺-ATPase and Mg²⁺ -pNPPase activities and steady-state Na⁺,-independent phosphorylation levels do not increase in parallel during development. These observations add further support to the proposed partial reaction scheme for (Na⁺- K⁺)-ATPase. The major increments in (Na ⁺-K⁺)-ATPase occur simultaneously with the deposition of specialized plasma membranes, particularly in the molecular layer of the cerebellum, as described in previous studies of rat hind brain.     

Harmaline: A competitive inhibitor of Na Ion in the (Na++K+)-ATPase system

Summary Experimental evidence is given that the hallucinogen harmaline (HME) behaves as an inhibitor of the (Na⁺+K⁺)-ATPase system, specifically in the Na⁺-dependent phosphorylation reaction. HME at 0.3 to 3mm inhibited several membrane ATPase preparations such as those from human erythrocytes, rat brain and squid retinal axons. The same concentration blocked Na⁺ outflow from squid giant axons. The behavior of several harmane derivatives such as harmine, harmalol and harmaline demonstrated that certain groups influenced the concentration for 50% inhibition of the ATPase system. The following evidence demonstrated that HME blocked the formation of the phosphorylated intermediate by competition with Na ions in the (Na⁺+K⁺)-ATPase reaction in rat brain. (1) The HME effect on the overall (Na⁺+K⁺)-ATPase reaction showed a fully competitive inhibition with respect to Na ion concentration. (2) The inhibition of the Na⁺-stimulated phosphorylation by HME was fully competitive with respect to Na ions, with or without oligomycin present. (3) HME inhibited the effect of ADP on the phosphorylation reaction using³²P-ATP. (4) HME did not accelerate the rate of membrane dephosphorylation by means of³²P-ATP and cold ATP.From the behavior of HME as a competitive inhibitor at Na ion sites of the (Na⁺+K⁺)-ATPase reactions one may gain information about (a) The chemical nature of Na⁺ sites which may be responsible for the selectivity of this cation, and (b) The sequence of Na⁺ and ATP entrance into the Na⁺-dependent phosphorylation reaction. The experimental evidence supports the hypothesis that the entrance of Na⁺ into the enzyme system may precede the formation of the phosphorylated intermediate.     

Irreversible zinc ion inhibition of (Na+ -K+)-adenosinetriphosphatase, Na+ -phosphorylation, and K+-p-nitrophenylphosphatase of Electrophorus electricus electroplax.

Zinc ion in micromolar concentrations is an irreversible inhibitor of Electrophorus electricus electroplax microsomal (Na⁺-K⁺)-ATPase. The rate of inhibition is dependent on [ZnCl₂] and the extent of inhibition varies with the ratio of ZnCl₂ to microsomal protein. The same kinetics are observed for inhibition of K⁺ -p-nitrophenylphosphatase and steady-state levels of Na⁺ -dependent enzyme phosphorylation. The observations suggest that a Zn²⁺ -sensitive conformational restraint is important to both kinase and phosphatase activities. The fact that inhibition is irreversible has implications for models seeking to relate zinc effects in tissue to inhibition of (Na⁺-K⁺)-ATPase.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

The role of glutathione in renal cortical tissue. effects of diamide on Na+ and GSSG levels,amino acid transport and Na+-K+-ATPase activity

Summary The effects of diamide were studied in rat kidney cortical tissue. It was found that diamide increased oxidized glutathione levels and inhibited Na⁺-K⁺-ATPase activity. Consistent with this finding was the observation that diamide compromised the sodium gradients maintained in renal cortical slices. Amino acid transport studies with ouabain or a sodium-free buffer indicated that diamide interferes with both Na⁺-dependent and Na⁺-independent transport systems. These results indicate that diamide has a number of different effects on renal cortical tissue and emphasize the important role of glutathione in maintaining control of a number of key metabolic pathways.     

Development of Na+ transport in the chicken colon

To evaluate the developmental changes in colonic Na⁺ transport, Na, K-ATPase activity and the sensitivity of the short-circuit current to amiloride were investigated. The amiloride-sensitive short-circuit current which represents the electrogenic, amiloride-sensitive Na⁺ transport through Na⁺ channels, was not present in chicken embryos but rose significantly after hatching in chicks which were kept on a low-salt diet. Amiloride-sensitive short-circuit current increased gradually but the plateau was not reached during the first 15 days of life. Drinking of 0.9% NaCl totally inhibited the induction of amiloride-sensitive Na⁺ transport. Na⁺, K⁺-ATPase activity increased during development but was not influenced by changes in salt intake. Na⁺ transport in chicken colon therefore undergoes profound developmental changes. The increase of Na⁺ transport refleets not only the adaptation of colonocytes to low salt intake but also the maturation of Na⁺ absorption in colon. The possible role of aldosterone in the adaptation to low-salt intake is discussed.Abbreviations LS low-salt - HS high-salt - I _sc short-circuit current     

Na+-transporting ATPase in the plasma membrane of halotolerant microalga Dunaliella maritima operates as a Na+ uniporter

A fraction of inside-out membrane vesicles enriched in plasma membranes (PM) was isolated from Dunaliella maritima cells. Attempts were made to reveal ATP-driven Na⁺-dependent H⁺ efflux from the PM vesicles to external medium, as detected by alkalization of the vesicle lumen. In parallel experiments, ATP-dependent Na⁺ uptake and electric potential generation in PM vesicles were investigated. The alkalization of the vesicle lumen was monitored with an impermeant pH-sensitive optical probe pyranine (8-hydroxy-1,3,6-pyrenetrisulfonic acid), which was loaded into vesicles during the isolation procedure. Sodium uptake was measured with ²²Na⁺ radioactive label. The generation of electric potential in PM vesicles (positive inside) was recorded with a voltage-sensitive probe oxonol VI. Appreciable Na⁺-and ATP-dependent alkalization of vesicle lumen was only observed in the presence of a protonophore CCCP (carbonyl cyanide-chlorophenylhydrazone). In parallel experiments, CCCP accelerated the ATP-dependent ²²Na⁺ uptake and abolished the electric potential generated by the Na⁺-ATPase at the vesicle membrane. A permeant anion NO^? ₃ accelerated ATP-dependent ²²Na⁺ uptake and promoted dissipation of the electric potential like CCCP did. At the same time, NO^? ₃ inhibited the ATP-and Na⁺-dependent alkalization of the vesicle lumen. The results clearly show that the ATP-and Na⁺-dependent H⁺ efflux from PM vesicles of D. maritima is driven by the electric potential generated at the vesicle membrane by the Na⁺-ATPase. Hence, the Na⁺-transporting ATPase of D. maritima carries only one ion species, i.e., Na⁺. Proton is not involved as a counter-ion in the catalytic cycle of this enzyme.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

Vanadate inhibition of rat cerebral cortex Na+, K+-ATPase during postnatal development

The Na⁺, K⁺-ATPase activity and its response to vanadate inhibition was investigated in cerebral cortex homogenates of 7-, 12- and 18-day-old rats. The enzyme was inhibited by vanadate in a dose-dependent manner in all these age groups. Furthermore, there was a different sensitivity towards vanadate during postnatal development; the concentration of V⁺⁵ needed for 50% inhibiton of Na⁺, K⁺-ATPase was 1.1×10^–6M, 2×10^–7M and 4.4×10^–7M for 7-, 12- and 18-day-old rats, respectively. It is suggested that the different sensitivity of Na⁺, K⁺-ATPase towards vanadate inhibition during postnatal development might be due to age-dependent changes in the ratio of various cell types.Special Issue dedicated to Dr. O. H. Lowry.     

Activation of Na+-K+-adenosine triphosphatase by spermine.

Spermine activated Na⁺-K⁺-ATPase when the concentrations of K⁺ and ATP were low, whereas it inhibited K⁺-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K⁺ or Na⁺. Excess K⁺ inhibited Na⁺-K⁺-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na⁺-K⁺-ATPase, and did not activate at all. It is suggested that the K⁺-sites essential to Na⁺-K⁺-ATPase and the K⁺-phosphatase co-exist at different places of the enzyme.     

LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH : I. Biochemical Characterization of Subcellular Fractions

In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na⁺, K⁺-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na⁺, K⁺-ATPase and ouabain insensitive Mg²⁺-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg²⁺-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na⁺, K⁺-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na⁺, K⁺-ATPase in M+L appeared to be associated with structures containing no Mg²⁺-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na⁺, K⁺-ATPase activity was highly dependent on the ratio of Na⁺ and K⁺ concentrations but independent of absolute concentrations over at least a fourfold range.     

Effects of prostaglandin A2 on Na+, K+-ATPase activity in basolateral plasma membrane of rat intestine in vitro

Prostagladin A₂, which prevents intestinal ulcers produced by administration of nonsteroidal antiinflammatory compounds such as indomethacin, inhibited the Na⁺,K⁺-ATPase activity in basolateral plasma membrane of rat intestine significantly. Prostaglandin A₂ inhibited mainly the Na⁺-dependent phosphorylation step in the overall reaction of Na⁺,K⁺-ATPase. This decrease of the Na⁺,K⁺-ATPase activity by prostaglandin A₂ was due to the decrease of Vmax of the enzyme and of the affinity of the enzyme for Na⁺. It was also suggested that the presence of both Δ^5,6 and Δ^10,11 structure of prostaglandin A₂ may be necessary for the inhibition of the Na⁺,K⁺-ATPase activity.     

Modification of the (Na+ + K+)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na⁺ + K⁺)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl (k_0.5 ca. 0.6 mm) or ATP (K_0.5 ca. 1 μm) in the medium protected against inactivation, whereas MgCl₂ (k_0.5 ca. 1 mm) increased inactivation. K⁺-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na⁺-dependent phosphorylation of the enzyme and Na⁺-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k_0.5 for K⁺-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k_0.5 for neither K⁺ nor Na⁺ was increased, although the apparent k_m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K⁺ at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k_m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a pK_a for the reactive enzyme groups of 7.5–8; and TNBS affected K⁺-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K⁺ at these sites.     

Purification,characterization and partial amino acid sequencing of a 70 kD inhibitor protein of Na+,K+-ATPase from goat testis cytosol

A protein isolated from goat testis cytosol is found to inhibit Na⁺,K⁺-ATPase from rat brain microsomes. The inhibitor has been purified by ammonium sulphate precipitation followed by hydroxyapatite column chromatography. The purified fraction appears as a single polypeptide band on 10% SDS-PAGE of approximate molecular mass of 70 kDa. The concentration at which 50% inhibition (I₅₀) occurs is in the nanomolar range. The inhibitor seems to bind Na⁺,K⁺-ATPase reversibly at ATP binding site in a competitive manner with ATP, but away from ouabain binding site. It does not affect p-nitrophenyl-phosphatase activity. The inhibitor is found to inhibit the phosphorylation step of the Na⁺,K⁺-ATPase. The enhancement of tryptophan fluorescence and changes in CD pattern suggest conformational changes of Na⁺,K⁺-ATPase on binding to the inhibitor. Amino acid sequence of the trypsinised fragments show some homology with aldehyde reductase.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Sensitivity and reversibility of Ca-dependent inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na⁺ + K⁺)-ATPase to inhibition by Ca was increased 30-fold by a partially purified extract of human red cell hemolysate. The hemolysate fraction reduced the concentration of free Ca required for 50% inhibition from 30 μM to approx. 1 μM. Ca-dependent inhibition of the (Na⁺ + K⁺)-ATPase in the presence and absence of the hemolysate fraction was completely reversible. The hemolysate fraction also stimulated the Ca²⁺-ATPase and increased its affinity for Ca. In the presence of the hemolysate fraction, the concentration of free Ca that inhibited the (Na⁺ + K⁺)-ATPase by 50% was similar to that which half-maximally stimulated the Ca²⁺-ATPase. Boiling the fraction destroyed its effect on the (Na⁺ + K⁺)-ATPase, but did not impair its stimulation of the Ca²⁺-ATPase.     

Quantitative calculation of the role of the Na+,K+-ATPase in thermogenesis

The Na⁺,K⁺-ATPase is accepted as an important source of heat generation (thermogenesis) in animals. Based on information gained on the kinetics of the enzyme's partial reactions we consider via computer simulation whether modifications to the function of the combined Na⁺,K⁺-ATPase/plasma membrane complex system could lead to an increased body temperature, either through the course of evolution or during an individual's lifespan. The enzyme's kinetics must be considered because it is the rate of heat generation which determines body temperature, not simply the amount of heat per enzymatic cycle. The results obtained indicate that a decrease in thermodynamic efficiency of the Na⁺,K⁺-ATPase, which could come about by Na⁺ substituting for K⁺ on the enzyme's extracellular face, could not account for increased thermogenesis. The only feasible mechanisms are an increase in the enzyme's expression level or an increase in its ion pumping activity. The major source of Na⁺,K⁺-ATPase-related thermogenesis (72% of heat production) is found to derive from passive Na⁺ diffusion into the cell, which counterbalances outward Na⁺ pumping to maintain a constant Na⁺ concentration gradient across the membrane. A simultaneous increase in both Na⁺,K⁺-ATPase activity and the membrane's passive Na⁺ permeability could promote a higher body temperature.