Cation transport coupled to ATP hydrolysis by the (Na, K)-ATPase

An integrated, animated model

Authors

  • Francisco A. Leone,

    Corresponding author
    1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brasil
    • Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto/Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brasil
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    • Tel.: +55 16 3602 3668; Fax: +55 16 3602 4838

  • Rosa P. M. Furriel,

    1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brasil
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  • John C. McNamara,

    1. Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brasil
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  • Jean D. Horisberger,

    1. Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland
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    • Deceased, July 1, 2009.

  • Ivana A. Borin

    1. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901 Ribeirão Preto, São Paulo, Brasil
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  • This work is supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Instituto Nacional de Ciência e Tecnologia (INCT) Adapta/Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM, No. 573976/2008-2)

Abstract

An Adobe® animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E1/E2-ATPase as it undergoes conformational changes between the E1 and E2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure–function relationship. The movements of the various domains within the (Na, K)-ATPase α-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the “core engine” of the pump, with respect to ATP binding, cation transport, and ADP and Pi release.

While Na+ and K+ ions can freely cross the cell membrane through specific ion channels in most animal cells, the cytosolic Na+ concentration is far lower than in the extracellular fluid in contrast to K+, which is much higher within the cell. This dual imbalance is maintained by the (Na, K)-ATPase that couples ATP hydrolysis to the quasi-simultaneous movement of both Na+ and K+ against their electrochemical potentials, thus, maintaining internally directed Na+ and externally directed K+ gradients across plasma membranes. Such gradients sustain important cell functions like osmotic equilibrium, volume regulation, pH homeostasis, membrane excitability, and secondary transport processes among many others [1, 2].

This transport protein is a heterodimer consisting of a 110-kDa α-subunit and a 55-kDa glycosylated β-subunit. As recently confirmed from the crystal structure of the (Na, K)-ATPase [3], the catalytic α-subunit exhibits 10 transmembrane α-helices that contain the cation binding sites. Together with the transmembrane segments, the cytoplasmic nucleotide binding (N), phosphorylation (P), and actuator (A) domains constitute the “core engine” of the ion pump, as also proposed for the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) [4]. The β-subunit, consisting of a single transmembrane span, is necessary for the functional expression and targeting of the α-subunit [5], and its possible influence on catalytic ion transport is an issue of ongoing discussion [6]. The kinetic properties of the enzyme also may be modulated via physical association with the so-called FXYD proteins, a family of small, homologous, single-span membrane proteins. To date, six members of this family are known to be associated with the (Na, K)-ATPase in a cell- or tissue-specific manner [7].

According to Axelsen and Palmgren's classification [8], the (Na, K)-ATPase is a P2c-type ATPase, that is, an ATP-powered ion pump that translocates cations across plasma membranes. These enzymes are also known as E1/E2-ATPases as they change their conformation between the E1 and E2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The two conformations differ with respect to ion affinities, the membrane leaflet from which the cation binding sites are accessible, sensitivity to ADP and ATP, intrinsic fluorescence, and sensitivity to proteolysis [4]. The energy required to move cations against their electrochemical gradients is derived from the hydrolysis of the terminal phosphate (γ-phosphate) bond of ATP, releasing phosphate and ADP.

During the catalytic cycle, an aspartate residue (D376) present in an invariant sequence DKTGT(I/L)T in such enzymes becomes phosphorylated, constituting the hallmark of the P-type ATPases [9]. According to the E1/E2 model [4], in the E1 state, the cation binding sites show high affinity for Na+ and are accessible from the cytoplasmic membrane leaflet. At this stage with ATP bound to the N domain, the α-subunit binds three Na+ ions, an event that induces a pronounced conformational change. The N domain rotates, positioning the ATP γ-phosphate close to the phosphorylation site of the P domain, allowing phosphorylation of the E1 form. Simultaneously, the A domain also rotates, releasing ADP, and the intracellular gate closes. The transformation of the high-energy E1-P to the E2-P form opens the extracellular gate leading to reconfiguration of the cation-binding sites. The release of the first Na+ ion to the extracellular fluid modifies the outer channel, accompanied by the release of the two remaining Na+ ions. The subsequent binding of two extracellular K+ ions to the empty cation-binding sites in the E2-P conformation catalyzes the dephosphorylation of D376, which closes the extracellular gate, occluding K+. This movement returns the N domain to its original position, exposing the empty nucleotide-binding site. The binding of a new ATP molecule promotes the transition from the E2(2K) to the E1 form, opening the intracellular gate and releasing the two K+ ions to the cytoplasm. A new cycle then proceeds.

A remarkable advance in the field of P-ATPase modeling has been the recent disclosure of the high-resolution structure of the SERCA [4, 10–12]. Although the degree of homology among the SERCA and the P2c-type ATPases varies considerably with regard to the complete amino acid sequence, the intracellular actuator and the nucleotide binding and phosphorylation domains are highly homologous, allowing the use of homology modeling to furnish credible structures for the P2c-type ATPases [13–20]. The complete crystal structure of the (Na, K)-ATPase from shark rectal gland is now known and corroborates these findings [21].

Here, we propose an animated model for use in undergraduate Biochemistry courses that accounts for the mechanism of ATP-hydrolysis coupled to cation transport by the (Na, K)-ATPase. The model illustrates in simplified, schematic form the movements and conformational changes occurring during the translocation of Na+ and K+ across the cell membrane, emphasizing the involvement of the A, N, and P domains, the “core engine” of this P2c-type ATPase, with respect to ATP binding, cation transport, and ADP and Pi release. While different P-ATPases may exhibit specific features defining the selectivity of their cation sites, their considerable homology suggests that observations pertaining to the SERCA can be extended to the (Na, K)-ATPase. However, for a more mechanistic view of the complex three-dimensional rotations and displacements of the cytoplasmic domains and transmembrane helices, original data available for the (Na, K)-ATPase must be examined [3, 21].

METHODS

The animated model was developed in Macromedia Flash 8.0® running on a Microsoft Windows® operating system, and is based on the scheme proposed by Horisberger [2] that considers recent significant findings regarding the (Na, K)-ATPase structure–function relationship [3, 21]. To build the animation, the graphics were created using drawing tools and stored in a library. Each graphic, selected from the library, was introduced at a chosen specific location in frames organized along a timeline, generating a logical sequence that reproduces the real chronological order of expected events. Finally, the finished frame sequence was compressed as a Macromedia Shockwave Flash file.

Most of the project was developed within a Windows® operating system environment. The integrated, animated (Na, K)-ATPase model (file name: Na, K-ATPase pumping cycle), provided in the Adobe SWF format, can be freely downloaded from http://portal.ffclrp.usp.br/sites/fdaleone/downloads. The package includes an automated installer; however, it is important to note that Adobe Flash Player must be installed before running the animation. Flash Player can be downloaded freely from http://www.adobe.com.

DESCRIPTION OF THE ANIMATED MODEL

Figure 1 shows a screenshot from the sodium–potassium pump animation. The catalytic cycle of the Na, K-ATPase begins with the enzyme in the El state (violet). As ATP occupies its binding site on the N domain (step E1ATP; blue), three Na+ ions (yellow) enter the transmembrane moiety of the α-subunit from the cytosol through the open intracellular gate (lower horizontal blue bar). The binding of these three ions to their high-affinity binding sites located within the transmembrane portion of the protein (step E1ATP3Na) produces the following conformational changes. Initially, the N domain undergoes extensive rotation, positioning the γ-phosphate of ATP close to the phosphorylation site. ATP is then hydrolyzed transferring the γ-phosphate to D376. After phosphorylation, the A domain (brown) also rotates resulting in a large translocation of the first transmembrane segment toward the intracellular leaflet. This movement leads to the closing of the intracellular gate (lower horizontal blue bar), occluding the three Na+ ions within the transmembrane segment of the (Na, K)-ATPase α-subunit, and is also accompanied by the release of ADP from the N domain (step E1P[3Na]). This constitutes the E1P state.

Figure 1.

Screenshot of the sodium–potassium pump cycle animation.

During the enzymatic cycle of the (Na, K)-ATPase, this high-energy E1P state rapidly decays to the E2P conformation (green). This change results in the opening of the extracellular gate (upper horizontal blue bar) with concomitant reconfiguration of the cation binding sites (step E2P3Na). A first Na+ ion is released, causing a large charge movement through the membrane (step E2P2Na) that is accompanied by reconfiguration of the outer access channel, causing the release of the other two Na+ ions (step E2P).

The cation-empty E2P conformation is now ready for the entrance of two extracellular K+ ions (magenta), the binding of which results in dephosphorylation of aspartate residue 376, and the concomitant closing of the extracellular gate (upper horizontal blue bar), occluding the two K+ ions within the transmembrane segment of the α-subunit (step E2[2K]).

The N domain is now accessible to intracellular ATP. During the catalytic cycle, ATP binding to the low-affinity intracellular site of the N domain (step E2ATP[2K]) induces the change to the E1 conformation (violet). This alteration includes opening the intracellular gate (lower horizontal blue bar) and the subsequent release of the two K+ ions to the intracellular fluid (step E1K). A new cycle then proceeds with the enzyme now in the E1ATP conformation.

As the animation proceeds, the different states assumed by the enzyme are indicated at the top of the screen. A pop-up key menu provides a key to the elements used in the animation, and a pop-up mechanism menu provides access to the reaction mechanisms of the catalytic cycle. Both pop-up menus can be opened or closed using their respective show or hide buttons. Although all the steps in the catalytic cycle are reversible, the animated model does not include the reverse reactions that may take place under appropriate conditions.

DISCUSSION

The use of animations in multimedia teaching and learning has increased considerably over the last few years, particularly owing to their presumed advantages over static graphics and tables. The use of animations for educational purposes often assumes that this method directs attention and increases interest in and motivation for a given issue, illustrates procedures, and generally explains how things work. However, learning with animations may be far less effective than is often supposed for several reasons. According to Lowe and Schnotz [22], the reduced effectiveness of multimedia applications generally results from the fact that animation design is not based on an understanding of what is required for learning from the animation. Corroborating this idea, Najjar [23] maintains that most current educational multimedia designs are based almost entirely on the opinions of experts rather than on empirical findings. Indeed, the educational effectiveness of a given animation depends on how its characteristics interact with the psychological functioning of the learner [22].

Although the model provided here illustrates in simplified, schematic form, the movements and conformational changes that occur during the translocation of Na+ and K+, across the membrane, coupled to ATP hydrolysis by the (Na, K)-ATPase, its value also lies in contemplating the most recent significant findings that have improved our understanding of the (Na, K)-ATPase structure–function relationship.

Finally, the model design itself considers several aspects valued by educators [23–25]. Blinking, scrolling, rapid movements, and continuous looping features were avoided. A single quick animation was generated to attract attention and not to distract the student. Simple controls were provided allowing the student to focus on the different steps of the catalytic cycle. As the animation is interactive, the student can control settings at any particular point in time of the animation. The animation was developed in Macromedia Flash 8.0® for the following reasons. Although Java, Maya, and Gif89 animations are compatible with the Windows, Macintosh, and Linux operating systems, we used Flash files as this application confers several benefits: Flash files are also compatible with Windows, Macintosh, and Linux; simple player controls can be introduced easily into the animation; Flash allows more complex interactivity; Flash files are smaller than video files; using Flash, illustrations and animations can be prepared in the same style.

CONCLUDING REMARKS

Animations focusing on various issues in biochemistry (e.g., conformational changes in proteins, ligand- or voltage-gated ion channels, protein motifs, and membrane structure) are generally viewed in an isolated fashion. The present animation, exploring the catalytic cycle of the sodium–potassium pump, is provided in an integrated manner, and as such can be used to examine other pertinent biochemical principles in the classroom. To illustrate: (i) alterations in protein conformation are linked to color changes (violet to green), allowing the student to observe how conformational changes come about without involving ruptures in protein structure; (ii) intrinsic membrane proteins (violet and green) embedded in the lipid bilayer; (iii) opening/closing of transient ligand-gated channels (sliding of upper and lower blue bars) as a function of Na+ or K+ binding; (iv) conformational changes (semisquares and semicircles in violet and green states, respectively) result in different Na+ and K+ binding affinities in the transmembrane moiety; (v) active countertransport of Na+ (yellow dots) and K+ (magenta squares); (vi) gradient-driven diffusion of Na+ and K+ through their respective ion channels across the lipid bilayer; and (vii) the integrated conformational movements of the A (brown), N (blue), and P (yellow) domains, site-specific ATP binding, and enzyme phosphorylation allow the student to visualize one of the most fundamental mechanisms of enzyme activity: regulation by phosphorylation/dephosphorylation.

Our animation has been used with rewarding results in many tasks over the past 4 years. It complements specific literature for scientific apprenticeship and master's and doctoral students developing research on the crustacean gill (Na, K)-ATPase. Using the animation, we focus on the structural and kinetic characteristics of the pump and its relevance to crustacean osmoregulation. We believe that the present animation is an efficient learning tool, allowing the undergraduate student to successfully visualize the active translocation of Na+ and K+ across the cell membrane during ATP hydrolysis by the (Na, K)-ATPase.

Acknowledgements

F. A. L and J. C. M. received research scholarships from CNPq.