1. Introduction

In the vast majority of cases, life has evolved in high-Na

⁺

environments [1], which are considered inadequate for, or injurious to, proper cellular functioning [2], notably without mechanisms that can maintain low intracellular Na

⁺

concentrations in organisms surrounded by and extracellular fluid containing ~150 mM Na

⁺

[3]. Thus, Na

⁺

extrusion from the intracellular milieu becomes a challenge and a prerequisite for the survival, growth, and the evolution of all species. Na

⁺

movement across the plasma membrane was initially described as being associated with K

⁺

movements. The first Na

⁺

pump described was (Na

⁺

+ K

⁺

)-ATPase. Two significant studies identified this pump: in the first, Skou [4] found that Na

⁺

and K

⁺

stimulated the catalysis of ATP hydrolysis by an ATPase in membrane fragments of leg nerves from the shore crab; in the second, Post & Jolly [5] demonstrated that Na

⁺

extrusion and K

⁺

uptake occur simultaneously in opposing directions and in an energy-dependent manner across the red-cell plasma membrane, with a stoichiometry of 3Na

⁺

:2K

⁺

.

A few years later, Proverbio et al. [6] described an ouabain-resistant Na

⁺

-ATPase activity that acts independently of K

⁺

in isolated membranes from the outermost cortex of a guinea pig, and that was sensitive. When they studied Na

⁺

fluxes through the basolateral membranes of proximal tubule cells towards the external milieu they found two mechanisms: (i) one coupled to K

⁺

and sensitive to ouabain, (ii) another one not coupled to K

⁺, resistant to ouabain and sensitive to ethacrynic acid ^[7]. More recently ^[8][9], the K

, resistant to ouabain and sensitive to ethacrynic acid [7]. More recently [8,9], the K

⁺

-independent and ouabain-resistant mechanism received the name of “second Na

⁺

pump”, which is still accepted [10].

The finding that the diuretic, furosemide, inhibits the ATP-dependent Na

⁺

efflux independently of K

⁺

(named for this reason Na

⁺

-ATPase) with no effect on the classic (Na

⁺

+ K

⁺

)-ATPase [11] was a significant step in identifying this Na

⁺

pump in several organisms. The main evolutionary advantage of a Na

⁺

-ATPase is its efficacy as a mechanism for Na

⁺

extrusion without interference with intracellular K

⁺ homeostasis. Rocafull et al. ^[9][12][13]purified and cloned the Na

homeostasis. Rocafull et al. [9,12,13] purified and cloned the Na

⁺

-ATPase from enterocytes (ATNA); its 3D structure was proposed, and the crucial amino acids of the catalytic and Na

⁺

binding sites were identified. They also demonstrated the enzyme’s sensitivity to furosemide and its resistance to ouabain. The existence of a second Na

⁺

pump—besides the (Na

⁺

+ K

⁺

)-ATPase—is now widely accepted.

The Na

⁺-ATPase from lower eukaryotes was first described in trypanosomatids ^[14][15][16]. In protozoan parasites, Na

-ATPase from lower eukaryotes was first described in trypanosomatids [14,15,16]. In protozoan parasites, Na

⁺

-ATPase, but not the (Na

⁺

+ K

⁺

)-ATPase, was found to be essential to energize the secondary active inorganic phosphate (P

_i) transport ^[17][18]. In apicomplexan parasites, Na

) transport [17,18]. In apicomplexan parasites, Na

⁺-ATPase may be a therapeutic target for malaria and toxoplasmosis ^[19][20].

-ATPase may be a therapeutic target for malaria and toxoplasmosis [19,20].

2. Trypanosomatid Parasites

The first evidence of a Na

⁺

-ATPase in a trypanosomatid parasite was presented by Caruso-Neves et al. [14], who showed that ouabain, the specific (Na

⁺

+ K

⁺

)-ATPase inhibitor, did not completely abolish Na

⁺

-stimulated ATPase activity of

Trypanosoma cruzi

epimastigotes, suggesting that this parasite has a Na

⁺

-ATPase that is insensitive to ouabain. Iizumi et al. [15] confirmed this observation and identified the protein. The gene responsible for Na

⁺

-ATPase in

T. cruzi

was named TcENA, due to its similarity to the gene for Na

⁺-ATPase from plants and fungi called ENA (ENA from exitus natru: the exit of sodium) ^[21][22]. TcENA (

-ATPase from plants and fungi called ENA (ENA from exitus natru: the exit of sodium) [21,22]. TcENA (

Figure 1

) has 10 possible transmembrane domains (TMpred Server), as well as for highly conserved domains corresponding to the Type-II P-ATPases catalytic sites: (1) the signature sequence DKTGT

³⁶⁵

containing aspartic acid that is phosphorylated during the catalytic cycle; (2) the domain DGFND

⁷⁰⁰

involved in Mg

²⁺

binding (green circle), and (3) the conserved TGEA

²⁰³

sequence (red circle), as well as F

⁴⁸⁰

, K

⁴⁸⁵

and K

⁵⁰⁴ (orange circle), which are related to the nucleotide-binding domain. These conserved domains are related to the Type IID P-type group; therefore, their presence in T. cruzi allowed TcENA to be included in the Type IID P-ATPases group ^[20], i.e., within a family of ion-transporting ATPases that form a phosphorylated intermediate during the catalytic cycle ^[23][24][25]. Phylogenetic analysis allowed for the inclusion of TcENA within the unique group Type IID or ENA-type P-ATPases, which includes

(orange circle), which are related to the nucleotide-binding domain. These conserved domains are related to the Type IID P-type group; therefore, their presence in T. cruzi allowed TcENA to be included in the Type IID P-ATPases group [20], i.e., within a family of ion-transporting ATPases that form a phosphorylated intermediate during the catalytic cycle [23,24,25]. Phylogenetic analysis allowed for the inclusion of TcENA within the unique group Type IID or ENA-type P-ATPases, which includes

Leishmania braziliensis

and

L. donovani

,

Saccharomyces cerevisiae

, and

Entamoeba histolytica

[20].

Figure 1.

Structural model of ENA ATPase from

T. cruzi

(TcENA). The model was constructed using the protein structure prediction PHYRE (

www.sbg.bio.ic.ac.uk/phyre/

) [26], based on the model of the SERCA ATPase and visualized with the standard molecular viewer PyMOL 2002 (DeLano, W.L. The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA, USA;

http://pymol.sourceforge.net/

). The 10 transmembrane helices are shown in orange, and the key residues mentioned in the text are highlighted by a red circle (conserved TGEA domain), an orange circle (F

⁴⁸⁰

, K

⁴⁸⁵

, K

⁵⁰⁴

residues related to nucleotide-binding); and a green circle (DGFND

⁷⁰⁰

domain involved in Mg

²⁺

binding).

T. cruzi

, the etiological agent of Chagas’ Disease, is a parasitic protozoan with a complex life cycle involving morphologically and functionally different stages that enable these parasites to adapt to a variety of conditions. In insect vectors, the proliferative epimastigotes form is found in the midgut, while in mammalian hosts, the gut contains the nonproliferative trypomastigote and the proliferative amastigote forms [27]. It may be that at some stages of the protozoan life cycle, different parasites—not only

T. cruzi

—would have different sensitivity to drugs that target Na

⁺

-ATPase.

A critical role for ATPases is to contribute to the maintenance of the plasma membrane potential (ΔΨ), which is the result of asymmetrical charge distribution when concentration gradients of H

⁺

, Na

⁺

, K

^+,

and Cl

⁻

are established in steady-state conditions. The ΔΨ of trypomastigotes forms is markedly sensitive to extracellular Na

⁺

and K

⁺

concentrations, and trypomastigotes have a high Na

⁺

-ATPase activity that contributes to a great extent in ΔΨ generation, in contrast with what happens in amastigotes. The Na

⁺

gradient in amastigote forms does not influence ΔΨ [28]. These observations match TcENA transcription levels, which vary among the different evolutionary forms of the parasite: trypomastigote and epimastigote forms have higher TcENA expression than amastigote forms [15]. Moreover, the subsequent addition of varying NaCl concentrations to a suspension of epimastigotes that were initially in Na

⁺

-free NMG medium (140 mM N-methylglucamine, 5 mM glucose, 1 mM MgSO

₄

, 1 mM CaCl

_2,

and 10 mM Hepes-Tris at pH 7.4) resulted in immediate and pronounced depolarization [28], consistent with the observation that Na

⁺

significantly stimulates ATPase activity of TcENA-expressing cells in a dose-dependent manner. Furthermore, epimastigotes overexpressing TcENA have salt tolerance, which is probably driven by their capacity for accelerated Na

⁺

efflux [15].