Computer simulation of synchronization of Na/K pump molecules

The behavior of Na/K pump currents when exposed to an oscillating electric field is studied by computer simulation. The pump current from a single pump molecule was sketched based on previous experimental results. The oscillating electric field is designed as a symmetric, dichotomous waveform varying the membrane potential from −30 to −150 mV around the membrane resting potential of −90 mV. Based on experimental results from skeletal muscle fibers, the energy needed to overcome the electrochemical potentials for the Na and K-transports are calculated in response to the field’s two half-cycles. We found that a specially designed oscillating electric field can eventually synchronize the pump molecules so that all the individual pumps run at the same pumping rate and phase as the field oscillation. They extrude Na ions during the positive half-cycle and pump in K ions during the negative half-cycle. The field can force the two ion-transports into the corresponding half-cycles, respectively, but cannot determine their detailed positions. In other words, the oscillating electric field can synchronize pumps in terms of their pumping loops but not at a specific step in the loop. These results are consistent with our experimental results in measurement of the pump currents.     

Evolution of the sodium pump

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na⁺ or H⁺ motive forces to drive Na⁺ and H⁺ dependent transport processes, respectively. For this purpose, animal rely on Na⁺/K⁺-ATPases whereas fungi and plants employ PM H⁺-ATPases. Prokaryotes, on the other hand, depend on H⁺ or Na⁺-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na⁺ and H⁺ pumps evolved? Here it is shown that prokaryotic Na⁺/K⁺-ATPases have near perfect conservation of binding sites involved in coordination of three Na⁺ and two K⁺ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H⁺-ATPases. With some exceptions, Na⁺/K⁺-ATPases and PM H⁺-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na⁺/K⁺-ATPases and PM H⁺-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H⁺ and Na⁺ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na⁺/K⁺-ATPases but lost PM H⁺-ATPases. At the same evolutionary branch point, fungi did loose Na⁺/K⁺-ATPases, and their role was taken over by PM H⁺-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na⁺/K⁺-ATPases but kept PM H⁺-ATPases.     

Autoregulation of the electrogenic sodium pump

The dependence of electrogenic sodium pump activity on changes in the cell volume of Helix pomatia neurons with different levels of intracellular sodium ion concentration was studied. Hypertonic solutions caused hyperpolarization of the membrane and increased membrane resistance in cells with a low sodium content (low-sodium cells; LSC). The activity of the electrogenic sodium pump in hypertonic solutions was increased compared to the activity in hypotonic solutions in LSC and decreased in cells with a high sodium content (high-sodium cells; HSC). The concentration of ouabain which led to maximal inhibition of active 22Na efflux from the neurons was 10(-4) M. Lower concentrations of ouabain (10(-8) M and lower) did not inhibit the sodium pump but stimulated it. The swelling of neurons in hypotonic solutions was accompanied by an increase in the number of binding sites for ouabain, while shrinking in hypertonic solutions led to the opposite effect--a decrease in binding sites. An increase in the number of binding sites also took place in normal isotonic potassium-free solutions compared with normal Ringer's solution. Two saturable components of ouabain binding were detectable in all solutions examined. gamma-Aminobutyric acid (GABA) and acetylcholine (ACh) increased the number of ouabain binding sites on the membrane. The results suggest that there are two opposite mechanisms by which cell volume changes can modulate the pump activity. One of them depends on the intracellular sodium ion concentration and causes pump activation in hypertonic solutions in LSC and saturation in HSC, while a second mechanism mediates the activating effect of cell swelling on the sodium pump in HSC. In addition, there may be a negative feedback between the pump activity and the number of functioning pump units in the membrane.     

Sulfatide role in the sodium pump

Summary Sodium efflux was studied in²²Na-loaded red blood cells in the presence of arylsulfatase, an enzyme that specifically hydrolyzes sulfatide. Sodium efflux was inhibited in proportion to the amount of arylsulfatase present. Maximum inhibition was almost as high as the efflux obtained in medium with K⁺ absent. At maximum inhibition 83.2% of the sulfatide content of the fragmented red blood cell membranes was hydrolyzed and ouabain-sensitive (Na⁺+K⁺)-ATPase activity was inhibited by 100%. Sodium efflux, sulfatide content, and (Na⁺+K⁺)-ATPase activity were unaffected with arylsulfatase in the presence of a high concentration of sulfatide. These results indicate that sulfatide plays a specific role in sodium and potassium ion transport. They also suggest that most sulfatide is localized externally in the red blood cell membrane.     

Computer simulation of flagellar movement. III. Models incorporating cross-bridge kinetics.

A computer simulation procedure is used to analyze the generation of propagated bending waves by flagellar models in which active sliding is generated by a cycle of cross-bridge activity. Two types of cross-bridge cycle have been examined in detail. In both cycles, cross-bridge attachment is followed immediately by a configurational change in the cross-bridge, which transfers energy to a stretched elastic element and generates a shearing force between the filaments. In the first model, which has cross-bridge behavior close to current ideas about cross-bridge behavior in muscle, cross-bridge attachment is proportional to curvature of the flagellum and detachment is an exponential decay process. The configurational change is equivalent to an angular deviation of pi/5 radians. In the second type of cross-bridge cycle, cross-bridge attachment occurs rapidly when a critical curvature is reached, and detachment occurs when a critical curvature in the opposite direction is reached. With this cycle, an unrealistically large angular deviation of the cross-bridges, equivalent to 3.0 radians, is required to obtain bending waves of normal amplitude. Both models generate bending wave patterns similar to those obtained in earlier work. However, the behavior of the second type of cross-bridge model more closely matches the actual behavior of flagella under experimental conditions: the chemical turnover rate per beat cycle remains constant as the viscosity is increased, and reduction in the number of active cross-bridges can cause a reduction in beat frequency, with little change in amplitude or wavelength.     

Proton diet for the sodium pump

In the absence of Na(+) and K(+) ions the Na,K-ATPase shows a pH-dependent ATP hydrolysis that can be inhibited by ouabain. At pH 7.2 this activity is 5% of the maximal under physiological conditions. It could be inferred that this activity is associated with H(+) transport in both directions across the membrane and facilitates an H-only mode of the sodium pump under such unphysiological conditions. By the analysis of experiments with reconstituted proteoliposomes an overall electroneutral transport mode has been proven. The stoichiometry was determined to be 2 H(+)/2 H(+)/1 ATP and is comparable to what is known from the closely related H,K-ATPase. By time-resolved ATP-concentration jump experiments it was found that at no time was the third, Na(+)-specific binding site of the pump occupied by protons. A modified Post-Albers pump cycle is proposed, with H(+) ions as congeners for Na(+) and K(+), by which all experiments performed can be explained.     

Vascular sodium pump activity kinetics in early and advanced stages of deoxycorticosterone-salt hypertension in rats.

We determined the kinetic properties--the maximal velocity, Vmax, and the half-maximal activating concentration of K+, km values--of the vascular sodium pump in rats 6, 28, and 50 days after deoxycorticosterone and sodium chloride (DOC-salt) or vehicle treatment. Tail arteries from six or eight rats from each treatment group were pooled, and Na-pump activity was measured in a Krebs medium containing varying K+ (plus 86Rb+) concentrations (0.25-10 mM). Na-pump activity was plotted as a function of [K + 86Rb]. Data were fit to a two-site model to calculate Vmax and km values. Systolic blood pressures were normal after 6 days but high after 28 and 50 days of DOC-salt treatment. No difference in kinetic parameters existed between the treatment and control groups 6 and 50 days after DOC-salt treatment. After 28 days, Vmax was significantly elevated compared with controls; km was not affected. Thus, stimulation of the vascular Na-pump during established hypertension is due to an increase in the maximal velocity of ouabain-sensitive uptake of K+.     

Mechanistic studies of sodium pump

Sodium pump was the first ion pump discovered. A member of the family of active transporters that catalyze adenosine 5′-triphosphate hydrolysis by forming a phosphorylated enzyme intermediate, sodium pump couples the energy released to unequal countertransport of sodium and potassium ions. The ion gradient generated by the pump is important for a variety of secondary physiological processes ranging from metabolite transport to electrical excitation of nerve and muscle. Selected experiments relating structure to function are reviewed.