How does reduced external K+ concentration affect the rate of Na+ efflux? Evidence against the K-Na coupled pump but in support of the association-induction hypothesis.

Recent frog-muscle studies produced the following findings: 1. Contrary to the theory of K+--Na+ coupled pump, reduction of external K+ concentration to near zero did not significantly reduce the rate of efflus of the fraction of cell Na+ conventionally regarded as rate-limited by membrane permeability. 2. Reduction of external K+ concentration profoundly reduced the rate of the efflux of this fraction only if the muscles were exposed to the low K+ while being loaded with radioactive Na+. 3. The data indicate that the fraction of Na+ efflux which in normal cells at room temperature has a half-time exchange (t1/2) of 20-40 min is not rate-limited by membrane permeability but by desorption from cellular adsorption sites. Surface-limited Na+ exchange between free Na+ in the cell and the external environment is represented by a faster fraction with a t1/2 of 2 to 4 min. 4. The data further indicate that the slow-down of the rate of efflux of the (slow) fraction arises from a cooperative shift of those beta- and gamma-carboxyl groups from adsorbing K+ to adsorbing Na+ when external K+ concentration is reduced below a critical level. The enhanced adsorption energy of the newly adsorbed Na+ raises the activation energy, hence a slower rate of exchange is seen as a slow-down in the "efflux curves." It is therefore only when free labeled Na+ is present in the cell water and thus available to the newly emerging Na+ adsorption sites that the effect of low external K+ can be visualized in a labeled-Na+ efflux study. Application of low K+ Ringer's solution after free labeled Na+ in and out of the cells has been washed away only causes enhanced adsorption of non-labeled Na+, which is not detected in isotope efflux study.     

Cellular functions of a cell in a metastable equilibrium state

A unifying hypothesis which might replace some of the many ion pumps which are invoked to describe distribution of ions across living cell membranes is developed quantitatively. Resting cells are assumed to be in a metastable state such that ions are in equilibrium between an extracellular aqueous phase, in which water has the properties of the bulk liquid, and an intracellular aqueous phase in which water has enhanced structure and strongly modified solvent properties. Partition coefficients or medium effects for Na⁺, K⁺ and Cl⁻ are calculated for several cell types. It is shown that in such a hypothetical cell, possessing no ion pumps there is an amplified Donnan potential between the two phases, its sign determined by the net charge on intracellular proteins, and its magnitude increased by a separation of ions induced by the difference in solvent properties of the water in the two phases. It is shown that a cell in such a metastable state is excitable and can generate an action potential with an inward surge of Na+ followed by an outward surge of K+. An explanation is offered for the transient release of Caa+ from the sarcoplasmic reticulum following excitation of a muscle fibre. Regulation of cellular volume is shown to be a necessary result of the presence in the extracellular solution of a high concentration of Na+, an ion with a very low affinity for intracellular water. It is concluded that the principal cellular functions that are commonly attributed to the sodium pump are also a feature of a cell in a metastable equilibrium state.     

Potassium retention in membraneless thymus lymphocyte nuclei

Nonionic detergents, Triton X-100 and Brij 58, removed lipoid membranes of suspended thymus lymphocytes within 5 minutes. The mobilization and solubilization of cytoplasmic and nuclear proteins occurred much faster (less than 5 minutes) with Triton X-100 treatment than with Brij 58 treatment (less than 10 minutes). In Triton X-100 treated cells the loss of K+ was complete within 5 minutes whereas with Brij 58 treatment the K+ loss was not complete after 10 minutes. Thus, the high concentration of K+ and the low concentration of Na+ in the nuclei can remain near normal for minutes in the absence of membrane structures. If the ions were in free solution within the cells, disruption of membrane integrity should lead to equilibration of the ions with external media within seconds. The decrease of K+ in the Brij 58 treated cells with exposure time was correlated with the solubilization of the proteins. These results support the view that K+ and Na+ are not freely dissolved in the cellular water, but are co-compartmentalized with proteins inside the living cell.     

The cellular resting and action potentials: interpretation based on the association-induction hypothesis

The Hodgkin, Huxley, and Katz theories of resting and action potentials are based on the membrane theory, which holds that cell K+ and water exist in the free state. Reviewed here are these theories of cellular potential along with the results of experimental testings. Reviewed also is Ling's association-induction (AI) hypothesis, which holds that all K+ is absorbed selectively and singly on anionic protein sites and that cell water is absorbed in multilayers on extended chains of "matrix proteins." In the development of the AI model, molecular mechanisms of cell permeation and electric potentials were presented according to which the potentials are surface-adsorption phenomena. Thus they resemble those suggested by Baur rather than the membrane potentials proposed by Ostwald and Bernstein. In the present review it is shown that the AI version of the surface adsorption model can account for evidence supporting the Hodgkin, Huxley, Katz approach as well as evidence against it-including extensive recent confirmation of the absorbed state of K+ in muscle.     

Na+/K+-ATPase activity of different types of striated muscles

Na+/K+-ATPase activity was determined in striated muscles with different aerobic capacities. The underlying hypothesis was that different aerobic capacities are reflective of different contractile activity which imposes greater demands on sarcolemmal ion translocation and may thus set Na pumping capacity. The added ion translocation demands required during exercise-training on Na+/K+-ATPase activity in different muscle fiber types may require an adaptation of this enzyme. The highest and lowest Na+/K+-ATPase activity was in the heart and white gastrocnemius muscle (WG), respectively. A high linear correlation existed between Na+/K+-ATPase activity and succinate dehydrogenase activity in the six muscles studied. Exercise-training did not increase Na+/K+-ATPase activity in any of the muscles, but did increase the aerobic capacity, except in the heart and WG. It was concluded that Na+/K+-ATPase activity has a high positive correlation with the aerobic capacity of striated muscles in the rat and that the Na pump capacity does not adapt to exercise-training of 1 hr X day-1 as does aerobic capacity.     

History of the membrane (pump) theory of the living cell from its beginning in mid-19th century to its disproof 45 years ago--though still taught worldwide today as established truth

The concept that the basic unit of all life, the cell, is a membrane-enclosed soup of (free) water, (free) K+ (and native) proteins is called the membrane theory. A careful examination of past records shows that this theory has no author in the true sense of the word. Rather, it grew mostly out of some mistaken ideas made by Theodor Schwann in his Cell Theory. (This is not to deny that there is a membrane theory with an authentic author but this authored membrane theory came later and is much more narrowly focussed and accordingly can at best be regarded as an offshoot of the broader and older membrane theory without an author.) However, there is no ambiguity on the demise of the membrane theory, which occurred more than 60 years ago, when a flood of converging evidence showed that the asymmetrical distribution of K+ and Na+ observed in virtually all living cells is not the result of the presence of a membrane barrier that permits some solutes like water and K+ to move in and out of the cell, while barring--absolutely and permanently--the passage of other solutes like Na+. To keep the membrane theory afloat, submicroscopic pumps were installed across the cell membrane to maintain, for example, the level of Na+ in the cell low and the level of K+ high by the ceaseless pumping activities at the expense of metabolic energy. Forty-five year ago this version of the membrane theory was also experimentally disproved. In spite of all these overwhelming evidence against the membrane-pump theory, it still is being taught as verified truth in all high-school and biology textbooks known to us today. Meanwhile, almost unnoticed, a new unifying theory of the living cell, called the association-induction hypothesis came into being some 40 years ago. Also little noticed was the fact that it has received extensive confirmation worldwide and has shown an ability to provide self-consistent interpretations of most if not all known experimental observations that are contradicting the membrane-pump theory as well as other observations that seem to support the membrane pump theory.     

Clearance of extracellular K+ during muscle contraction--roles of membrane transport and diffusion

Excitation of muscle often leads to a net loss of cellular K + and a rise in extracellular K+([K+]o), which in turn inhibits excitability and contractility. It is important, therefore, to determine how this K+ is cleared by diffusion into the surroundings or by reaccumulation into the muscle cells. The inhibitory effects of the rise in [K+] o may be assessed from the time course of changes in tetanic force in isolated muscles where diffusional clearance of K+ is eliminated by removing the incubation medium and allowing the muscles to contract in air. Measurements of tetanic force, endurance, and force recovery showed that in rat soleus and extensor digitorum longus (EDL) muscles there was no significant difference between the performance of muscles contracting in buffer or in air for up to 8 min. Ouabain-induced inhibition of K+ clearance via the Na+,K+ pumps markedly reduced contractile endurance and force recovery in air. Incubation in buffer containing 10 mM K+ clearly inhibited force development and endurance,and these effects were considerably reduced by stimulating Na+,K+ pumps with the 2 -agonist salbutamol. Following 30-60 s of continuous stimulation at 60 Hz, the amount of K + released into the extracellular space was assessed from washout experiments. The release of intracellular K+ per pulse was fourfold larger in EDL than in soleus,and in the two muscles, the average [K+] o reached 52.4 and 26.0 mM, respectively, appreciably higher than previously detected. In conclusion, prevention of diffusion of K+ from the extracellular space of isolated working muscles causes only modest interference with contractile performance. The Na+,K+ pumps play a major role in the clearance of K+ and the maintenance of force. This new information is important for the evaluation of K+ -induced inhibition in muscles, where diffusional clearance of K+ is reduced by tension development sufficient to suppress circulation.     

Insights into the biochemistry of the ubiquitous NhaP family of cation/H+ antiporters

Na+/H+ antiporters are integral membrane proteins that exchange Na+ for H+ across the cytoplasmic or organellar membranes of virtually all living cells. They are essential for control of cellular pH, volume homeostasis, and regulation of Na+ levels. Na+/H+ antiporters have become increasingly characterized and are now becoming important drug targets. The recently identified NhaP family of Na+/H+ antiporters, from the CPA1 superfamily, contains proteins with a surprisingly broad collective range of transported cations, exchanging protons for alkali cations such as Na+, Li+, K+, or Rb+ as well as for Ca2+ and, possibly, NH4+. Questions about ion selectivity and the physiological impact of each particular NhaP antiporter are far from trivial. For example, Vc-NhaP2 from Vibrio cholerae has recently been shown to function in vivo as a specific K+/H+ antiporter while retaining the ability to exchange H+ for Na+ and bind (but not exchange with H+) Li+ in a competitive manner. These and other findings reviewed in this communication make antiporters of the NhaP type attractive systems to study intimate molecular mechanisms of cation exchange. In an evolutionary perspective, the NhaP family seems to be a phylogenetic entity undergoing active divergent evolution. In this minireview, to rationalize peculiarities of the cation specificity in the NhaP family, the "size-exclusion principle" and the idea of "ligand shading" are discussed.     

Na+-Ca2+ exchanger in the soluble fraction of crayfish striated muscle

Proteins with Na+-Ca2+ exchange activity from the soluble fraction of crayfish striated muscle were inserted into asolectin proteoliposomes. A pH dependent calcium uptake with an optimum at the alkaline side and inhibition in the presence of sodium or strontium ions in the external medium was observed. When expressed per tissue wet weight the capacity for Na+-Ca2+ exchange of proteoliposomes with inserted soluble proteins was by one half higher than that of the membrane fraction and more than twice higher in comparison with the reconstituted membrane bound exchanger. Using polyacrylamide gel electrophoresis two most prominent proteins with Mr over 200 and 43 kDa could be detected in proteoliposomes with the highest Na+-Ca2+ exchange. It is assumed that protein(s) with Mr 43 kDa could represent the soluble Na+-Ca2+ exchanger in crayfish striated muscle soluble fraction.     

Thallium and cesium in muscle cells compete for the adsorption sites normally occupied by K+

It was shown that Tl+ can stoichiometrically displace K+ in frog muscle cells. This displacement is independent of the function of cell membrane and postulated pumps and of a macroscopic electric charge or Donnan effect. It follows that Cs+ and Tl+ compete for the adsorption sites normally occupied by K+. On this basis, the association-induction hypothesis predicts that Cs+ and Tl+, like K+, should be primarily localized in the A-band of the muscle myofibrils.     

Thymocyte K+, Na+ and water balance during dexamethasone- and etoposide-induced apoptosis.

The mechanism of apoptotic cell volume decrease was studied in rat thymocytes treated with dexamethasone (Dex) or etoposide (Eto). Cell shrinkage, i.e. dehydration, was quantified by using buoyant density of the thymocytes in a continuous Percoll gradient. The K+ and Na+ content of cells from different density fractions were assayed by flame emission analysis. Apoptosis was tested by microscopy and flow cytometry of acridine orange stained cells as well as by flow DNA cytometry. Treatment of the thymocytes with 1 microM Dex for 4-5.5 h or 50 microM Eto for 5 h resulted in the appearance of a new distinct high-density cell subpopulation. The cells from this heavy subpopulation but not those with normal buoyant density had typical features of apoptosis. Apoptotic increase of cell density was accompanied by a decrease in cellular K+ content, which exceeded the simultaneous increase in cellular Na+ content. Cellular loss of K+ contributed to most of the estimated loss of cellular osmolytes, but owing to the parallel loss of cell water, the decrease in cytosolic K+ concentration was less than one third. Due to gain of Na+ and loss of cell water the cytosolic Na+ concentration in thymocytes rose following treatment with Dex (5.5 h) or Eto (5 h) by a factor of about 3.6 and 3.1, respectively.     

Na+-Ca2+ exchange in plasma membranes of crayfish striated muscle

Na+-Ca2+ exchange rates and some physico-chemical properties of the exchanger were studied in crayfish striated muscle membranes enriched in plasma membranes prepared by differential centrifugation of muscle microsomal fraction on discontinuous sucrose density gradient. The lightest subfraction with the highest Na+, K+-ATPase and Mg2+-ATPase activities also showed the highest Na+-Ca2+ exchange rates. A number of physico-chemical characteristics of the Na+-Ca2+ exchanger found in the present experiments were similar to those reported for excitable membranes of mammals, except for the temperature optimum (20 degrees C for the crayfish).     

A cooperative transition theory applied to the kinetics of ionic exchanges in cells

Manifestations of a cooperative interaction between ion-adsorbing sites in cells include steep, sigmoidal equilibrium adsorption isotherms of K+ and Na+, critical temperature transitions of net exchanges of Na+ for K+, and the allosteric nature of the effects of ligands on cellular K+ and Na+. Cooperative ionic adsorption is described by a one-dimensional Ising model. The experimentally-determined equilibrium parameters permit prediction of the kinetics of exchange of K+ for Na+ (the approach to equilibrium) by stochastic or hydrodynamic solutions of a time-dependent Ising model. Studies of the rates of self-exchange of adsorbed ions reveal properties of the cooperatively interacting adsorption sites and their dependence on temperature and chemical potential. High rates of isotopic exchanges of K+ and Na+ occur near the transition point. This is explained by the hypothesis of an increase in susceptibility of the ensemble to slight variations of microK or microNA near the phase transition, which leads to an increase in microscopic fluctuations within the ensemble. It is suggested that the isotopic ion exchange experiment may be a means to explore the microscopic states of the ensemble and their transition probabilities.     

Kinetic investigation of K+ efflux during glycerol treatment of muscle

The kinetics of K+ efflux was investigated in the membranes of frog sartorius muscle after disintegration by glycerol treatment. Data of the K+ concentration in the muscle as a function of time of the glycerol treatment fitted well the sum of two exponential fractions (with the correlation coefficient of more than 0.98). The half-lives of the two fractions of the K+ efflux were 1 and 75 hours respectively. On the basis of the value of its half life the efflux of the faster fraction was suggested to correspond to the free diffusion. At low temperature the magnitude of the faster fraction increased in a Na+-containing milieu. This could be due to K+-Na+ ion exchange. From the rate of loss of the slower fraction of K+ one finds that movement of K+ in cells without membranes is significantly slower than free diffusion. Presumably, part of the bound potassium exists in intra- or intermolecular "ion-bridges" of muscle proteins.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Active sodium transport in basolateral plasma membrane vesicles from rat kidney proximal tubular cells

Inside-out vesicles prepared with basolateral plasma membranes from rat kidney proximal tubular cells can accumulate Na+ actively in two ways. Mode 1, which is K+-independent, is ouabain-insensitive and is inhibited by furosemide and mode 2, which is K+-dependent, is inhibited by ouabain and is insensitive to furosemide. The presence of Mg2+ and ATP in the incubation medium is essential for both modes of Na+ uptake to proceed and in both cases, the nucleotide is hydrolyzed during the process. These results are consistent with the idea of the existence, in these membranes, of two Na+ pumps: one, which can work in the absence of K+ (Na+ pump) and another, which needs K+ to work (Na+ + K+ pump).     

Role of the calpain system in muscle growth.

Muscle protein degradation has an important role in rate of muscle growth. It has been difficult to develop procedures for measuring rate of muscle protein degradation in living animals, and most studies have used in vitro systems and muscle strips to determine rate of protein degradation. The relationship between results obtained by using muscle strips and rate of muscle protein turnover in living animals is unclear because these strips are in negative nitrogen balance and often develop hypoxic cores. Also, rate of protein degradation is usually estimated by release of labeled amino acids, which reflects an average rate of degradation of all cellular proteins and does not distinguish between rates of degradation of different groups of proteins such as the sarcoplasmic and the myofibrillar proteins in muscle. A number of studies have suggested that the calpain system initiates turnover of myofibrillar proteins, which are the major group of proteins in striated muscle, by making specific cleavages that release thick and thin filaments from the surface of the myofibril and large polypeptide fragments from some of the other myofibrillar proteins. The calpains do not degrade myofibrillar proteins to small peptides or to amino acids, and they cause no bulk degradation of sarcoplasmic proteins. Hence, the calpains are not directly responsible for release of amino acids during muscle protein turnover. Activity of the calpains in living cells is regulated by calpastatin and Ca2+, but the nature of this regulation is still unclear.     

Na(+)-Ca2+ exchange in locust striated muscles

High Na+ + Ca2+ exchange rates comparable with those reported for crayfish striated muscle, rat heart and rat brain, were observed in locust striated muscle homogenates and membrane preparations. The Na(+)-Ca2+ exchange followed the 1st order kinetics with a Km value of 18 mumol.l-1 for Ca, the pH optimum was at 8, the temperature optimum at 30 degrees C, and the exchange was inhibited in the presence of sodium in the incubation medium, with a KiNa of approx. 25 mmol.l-1. The present results suggest a high Na(+)-Ca2+ exchange in locust striated muscles which operate on the calcium electrogenesis principle.     

Identification of uterine ion transporters for mineralisation precursors of the avian eggshell

ABSTRACT: BACKGROUND: In Gallus gallus, eggshell formation takes place daily in the hen uterus and requires large amounts of the ionic precursors for calcium carbonate (CaCO3). Both elements (Ca2+, HCO3-) are supplied by the blood via trans-epithelial transport. Our aims were to identify genes coding for ion transporters that are upregulated in the uterine portion of the oviduct during eggshell calcification, compared to other tissues and other physiological states, and incorporate these proteins into a general model for mineral transfer across the tubular gland cells during eggshell formation. RESULTS: A total of 37 candidate ion transport genes were selected from our database of overexpressed uterine genes associated with eggshell calcification, and by analogy with mammalian transporters. Their uterine expression was compared by qRTPCR in the presence and absence of eggshell formation, and with relative expression levels in magnum (low Ca2+/HCO3- movement) and duodenum (high rates of Ca2+/HCO3- trans-epithelial transfer). We identified overexpression of eleven genes related to calcium movement: the TRPV6 Ca2+ channel (basolateral uptake of Ca2+), 28 kDa calbindin (intracellular Ca2+ buffering), the endoplasmic reticulum type 2 and 3 Ca2+ pumps (ER uptake), and the inositol trisphosphate receptors type 1, 2 and 3 (ER release). Ca2+ movement across the apical membrane likely involves membrane Ca2+ pumps and Ca2+/Na+ exchangers. Our data suggests that Na+ transport involved the SCNN1 channel and the Na+/Ca2+ exchangers SLC8A1, 3 for cell uptake, the Na+/K+ ATPase for cell output. K+ uptake resulted from the Na+/K+ ATPase, and its output from the K+ channels (KCNJ2, 15, 16 and KCNMA1).We propose that the HCO3- is mainly produced from CO2 by the carbonic anhydrase 2 (CA2) and that HCO3- is secreted through the HCO3-/Cl- exchanger SLC26A9. HCO3- synthesis and precipitation with Ca2+ produce two H+. Protons are absorbed via the membrane's Ca2+ pumps ATP2B1, 2 in the apical membrane and the vacuolar (H+)-atpases at the basolateral level. Our model incorporate Cl- ions which are absorbed by the HCO3-/Cl- exchanger SLC26A9 and by Cl- channels (CLCN2, CFTR) and might be extruded by Cl-/H+ exchanger (CLCN5), but also by Na+ K+ 2 Cl- and K+ Cl- cotransporters. CONCLUSIONS: Our Gallus gallus uterine model proposes a large list of ion transfer proteins supplying Ca2+ and HCO3- and maintaining cellular ionic homeostasis. This avian model should contribute towards understanding the mechanisms and regulation for ionic precursors of CaCO3, and provide insight in other species where epithelia transport large amount of calcium or bicarbonate.     

Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half- saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half- maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.