Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin

doi:10.1016/j.compbiolchem.2007.08.001

Computational Biology and Chemistry

Volume 32, Issue 1, February 2008, Pages 5-16

https://doi.org/10.1016/j.compbiolchem.2007.08.001 Get rights and content

Abstract

The ATP hydrolysis reactions responsible for the Na⁺/K⁺-ATPase phosphorylation, according to recent experimental evidences, also occur for the PTX–Na⁺/K⁺ pump complex. Moreover, it has been demonstrated that PTX interferes with the enzymes phosphorylation status. However, the reactions involved in the PTX–Na⁺/K⁺ pump complex phosphorylation are not very well established yet. This work aims at proposing a reaction model for PTX–Na⁺/K⁺ pump complex, with similar structure to the Albers–Post model, to contribute to elucidate the PTX effect over Na⁺/K⁺-ATPase phosphorylation and dephosphorylation. Computational simulations with the proposed model support several hypotheses and also suggest: (i) phosphorylation promotes an increase of the open probability of induced channels; (ii) PTX reduces the Na⁺/K⁺ pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na⁺/K⁺-ATPase may not be phosphorylated; (iv) PTX can bind to substates of the two principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity sites. The proposed model also allows previewing the behavior of the PTX–pump complex substates for different levels of intracellular ATP concentrations.

Introduction

The basic requirements for the cellular activity are the ionic gradients through the membrane. These gradients provide energy for several essential cellular functions like cell volume regulation and for secondary active transports of amino acids, sugars, bile acids, neurotransmitters, ions, and other solutes across the cell boundary. The Na⁺/K⁺-ATPase, currently known as Na⁺/K⁺ pump or sodium pump, is responsible for establishing and maintaining these electrochemical gradients in animal cells. The Na⁺/K⁺ pump is a plasma membrane-bound protein that exchanges three Na⁺, from the intracellular space, and two K⁺, from the external medium, across the membrane, at the expenditure of energy derived from the ATP splitting (Post et al., 1972, Rakowski et al., 1989; Cornelius, 1999).

Nowadays, the understating of the Na⁺/K⁺ pump kinetics is outlined in the alternating-gate model of Albers–Post transport cycle (Post et al., 1965, Post et al., 1972, Albers et al., 1963, Taniguchi and Post, 1975, Läuger, 1979). According to such a model, the Na⁺ and K⁺ exchange results from reactions between these ions and the Na⁺/K⁺-ATPase. Briefly, an internal and an external gate, one on each side of the ion-binding cavity, are supposed to exist and these gates are constrained to open and close alternately. The Na⁺/K⁺-ATPase alternates between two principal conformational states: E1 and E2. At the first state, with the intracellular gate opened and ATP present in its binding site, the entry of three Na⁺ and the binding of these ions in their high-affinity sites induce the ATP hydrolyses promoting occlusion of the Na⁺ ions. Spontaneously, this high-energy E1 state rapidly changes to the E2 with the opening of the extracellular gate, the release of Na⁺ ions, the entry of two K⁺ and the binding to their high-affinity sites. The two K⁺ binding causes dephosphorylation and the extracellular gate closing. The release of these two K⁺ into the intracellular space completes the pump cycle.

Experiments conducted to study the interference of a toxin, called palytoxin (PTX), with the pump activity showed that this toxin induces a dissipative flux of monovalent ions through the cellular membrane (Chhatwal et al., 1983, Grell et al., 1988, Muramatsu et al., 1988). From these results, the prediction that PTX would be able to promote the uncoupling between the external and internal pump gates was stated (Habermann, 1989, Scheiner-Bobis and Schneider, 1997, Scheiner-Bobis, 2002, Artigas and Gadsby, 2003b, Artigas and Gadsby, 2004, Horisberger, 2004). Additionally, it has also been shown that the PTX–pump interactions are dependent on the enzyme phosphorylated and dephosphorylated states (Artigas and Gadsby, 2002, Artigas and Gadsby, 2003a, Artigas and Gadsby, 2003b, Artigas and Gadsby, 2004, Tosteson et al., 2003). Nevertheless, the chemical reactions involved in the PTX-bound pump phosphorylation are not yet very well established and it is reasonable to expect that these reactions constitute a complex system.

In this paper, we endeavor to propose an Albers–Post type reaction scheme to investigate how PTX interferes with the enzyme phosphorylation and dephosphorylation. The computer simulations of measurements of Artigas and Gadsby, 2003a, Artigas and Gadsby, 2004 and Tosteson et al. (2003) and the subsequent analysis of the reactions kinetics involved support several hypotheses and also suggest: (i) phosphorylation increases the open probability of the PTX-induced channels; (ii) PTX reduces the Na/K pump phosphorylation rate; (iii) PTX may cause conformational changes to substates where the Na/K-ATPase may not be phosphorylated; (iv) PTX can bind to substates from the principal states E1 and E2, with highest affinity to phosphorylated enzymes and with ATP bound to its low-affinity site. Moreover, the proposed model allows previewing the behavior of the PTX–pump complex substates for different levels of intracellular ATP concentrations.

Section snippets

Model

The reaction model for the phosphorylation and dephosphorylation of the PTX–pump complex was developed adding to the Albers–Post model reactions that describe substates involving the PTX.

Effect of PTX on the Enzyme Phosphorylation

Investigating the PTX effect over the Na⁺/K⁺-ATPase phosphorylation by ATP, Tosteson et al. (2003) observed that the amount of the phosphorylated form of the enzyme is reduced when exposed to PTX. This fact suggests that the PTX binding blocks the ATP binding or stimulates P unbinding or both. Since in this experiment the [ATP]ⁱ was very reduced, ≈10 μM, the ATP low-affinity binding site occupation, according to Campos and Beaugé (1994), would not be significant. Additionally, before the ATP

Discussion

The main goal of the present work is to propose a reaction model for PTX–pump complex to simulate and contribute to the elucidation of the PTX effect over Na⁺/K⁺-ATPase phosphorylation and dephosphorylation. The reactions follow the same structure of the Albers–Post model and were deduced analyzing and simulating experiments of Artigas and Gadsby, 2003a, Artigas and Gadsby, 2004 and Tosteson et al. (2003). Supporting the hypothesis proposed by Artigas and Gadsby (2004), the simulations

Acknowledgements

This work was supported by FAPEMIG, CNPq and PROCAD/CAPES Foundation.

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Model and simulation of Na+/K+ pump phosphorylation in the presence of palytoxin

Abstract

Introduction

Section snippets

Model

Effect of PTX on the Enzyme Phosphorylation

Discussion

Acknowledgements

J. Biol. Chem.

Biophys. J.

Toxicon

J Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

The role of sodium ions in the activation of electrophorys electric organ adenosine trisphosphatase

Biochemistry

Ion channel-like properties of the Na+/K+ pump

Ann. NY Acad. Sci.

Na+/K+-pump ligands modulate gating of palytoxin ion channels

Proc. Natl. Acad. Sci. U.S.A.

Ion occlusion/deocclusion partial reactions in individual palytoxin-modified Na/K pumps

Proc. Natl. Acad. Sci U.S.A.

Model and simulation of Na⁺/K⁺ pump phosphorylation in the presence of palytoxin

Ion channel-like properties of the Na⁺/K⁺ pump

Na⁺/K⁺-pump ligands modulate gating of palytoxin ion channels