Palytoxin-induced Effects on Partial Reactions of the Na,K-ATPase

The interaction of palytoxin with the Na,K-ATPase was studied by the electrochromic styryl dye RH421, which monitors the amount of ions in the membrane domain of the pump. The toxin affected the pump function in the state P-E2, independently of the type of phosphorylation (ATP or inorganic phosphate). The palytoxin-induced modification of the protein consisted of two steps: toxin binding and a subsequent conformational change into a transmembrane ion channel. At 20°C, the rate-limiting reaction had a forward rate constant of 105 M−1s−1 and a backward rate constant of about 10−3 s−1. In the palytoxin-modified state, the binding affinity for Na+ and H+ was increased and reached values between those obtained in the E1 and P-E2 conformation under physiological conditions. Even under saturating palytoxin concentrations, the ATPase activity was not completely inhibited. In the Na/K mode, ∼50% of the enzyme remained active in the average, and in the Na-only mode 25%. The experimental findings indicate that an additional exit from the inhibited state exists. An obvious reaction pathway is a slow dephosphorylation of the palytoxin-inhibited state with a time constant of ∼100 s. Analysis of the effect of blockers of the extracellular and cytoplasmic access channels, TPA+ and Br2-Titu3+, respectively, showed that both access channels are part of the ion pathway in the palytoxin-modified protein. All experiments can be explained by an extension of the Post-Albers cycle, in which three additional states were added that branch off in the P-E2 state and lead to states in which the open-channel conformation is introduced and returns into the pump cycle in the occluded E2 state. The previously suggested molecular model for the channel state of the Na,K-ATPase as a conformation in which both gates between binding sites and aqueous phases are simultaneously in their open state is supported by this study

Finding Na,K-ATPase II - From fluxes to ion movements

After identification of the Na,K-ATPase as active ion transporter that maintains the Na+ and K+ concentration gradient across the membrane of virtually all animal cells, a long history of mechanistic studies began in which enzyme activity and ion-transport were intensively investigated. A basis for detailed understanding was laid in the so-called Post-Albers pump cycle. Developing new experimental techniques allowed the determination of different flux modes, the analysis of the kinetics of enzyme phosphorylation and dephosphorylation as well as of the transport of Na+ and K+ ions across the membrane. The accumulation of results from transport studies allowed the proposal of the gated channel concept that turned out to be a successful approach to explain the transport-related experimental findings. Eventually, it found its counterpart in the high-resolution structure of the ion pump. Recently it turned out that simple mutations of the Na,K-ATPase are the cause of several diseases

Finding Na,K-ATPase: I - From Cell to Molecule

The oppositely oriented concentration gradients of Na+ and K+ ions across the cell membrane as found in animal cells led to the requirement of an active ion-transport mechanism that maintains this steady-state condition. As solution of this problem the Na,K-ATPase was identified, a member of the P-type ATPase family. Its stoichiometry has been defined as 3 Na+/2 K+/1 ATP, and a class of Na,K-ATPase-specific inhibitors, cardiac steroids, was established, which allow the identification of this ion pump. In an effort lasting for several decades structural details were uncovered down to almost atomic resolution. The quaternary structure of the functional unit, either αβ heterodimer or (αβ)n complexes with n ≥ 2, is still under discussion

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

AbstractThe 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

Subtle mutation, far-reaching effects

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Mechanistic Principles of Ion Transport in the Na,K-ATPase

The Na,K-ATPase is a member of the P-type ATPase family and a primary active ion transporter for Na+ and K+ ions in the cytoplasmic membrane of virtually all animal cells. Considerable progress in understanding the ion-pump mechanism of the Na,K-ATPase was gained by combining biophysical and biochemical studies of more than 30 years with structural information at atomic resolution available since recent years. Biophysical studies have revealed detailed properties of the ion movements that led to a gated-channel model which is strongly supported by structural findings obtained for the sodium pump. The basic question how the free Gibbs energy released by ATP hydrolysis is transferred to the protein and transformed into uphill transport of the ions is still without reply.publishe

How do P-Type ATPases transport ions?

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins, the energy-providing ATP hydrolysis is coupled to ion transport of one or two ion species across the respective membrane. The pump function of the investigated pumps is described by a so-called Post-Albers cycle. Main features of the pumping process are (1) a Ping-Pong mechanism, i.e. both transported ion species are transferred successively and in opposite direction across the membrane, (2) the transport process for each ion species consists of a sequence of reaction steps, which are ion binding, ion occlusion, conformational transition of the protein, successive deocclusion of the ions and release to the other side of the membrane. (3) Recent experimental evidence shows that the ion-binding sites are placed in the transmembrane section of the proteins and that ion movements occur preferentially during the ion binding and release processes. The main features of the mechanism include narrow access channels from both sides, one gate per access channel, and an ion-binding moiety that is adapted specifically to the ions that are transported, and differently in both principal conformations

Toward an Understanding of Ion Transport through the Na,K-ATPase

In the Na,K-ATPase the charge-translocating reaction steps were found to be binding of the third Na+ ion to the cytoplasmic side and the release of all three Na+ ions to the extracellular side as well as binding of the two K+ ions on the extracellular side. The conformation transition E1 -> E2 was only of minor electrogenicity; all other reaction steps produced no significant charge movements. In the SR Ca-ATPase and the gastric H,K-ATPase, all ionbinding and -release steps were identified to move charge through the membrane. The high-resolution structure of the SR Ca-ATPase in state E1 revealed the position of the ion-binding sites in the transmembrane part of the protein. If the same arrangement is assumed for the Na pump, the missing expected charge movements in state E1 may to be assumed to be apparent effects. With the proposal that binding of 2 Na+ or 2 K+ is compensated correspondingly by H+ ions, agreement between structural and functional aspects is obtained. Investigations of the pH-dependence of ion-binding steps indicate competition between the ions and electrogenic H+ binding in support of this concept