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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ClC chloride channels perform a wide variety of physiological functions and they had been characterized in animals, yeast, plants and bacteria but not in protozoa. By blast search we found in Entamoeba histolytica, the protozoan responsible for human amoebiasis, two genes (Ehclc-A and Ehclc-B) encoding for putative polypeptides with 25–30% identity to ClC chloride channels of several organisms. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that both genes are transcribed in trophozoites. Phylogenetic analysis revealed that EhClC-A and EhClC-B polypeptides belong to the eukaryotic branch of plasma membrane ClCs. Specific antibodies against EhClC-A confirmed that it is located at the trophozoite plasma membrane. Xenopus laevis oocytes microinjected with Ehclc-A cRNA elicited anion currents not detected in oocytes microinjected with water. Induced currents were inwardly rectifying and had a permeability sequence of Cl > Br > I > F >> NO3. The chloride channel blocker 4-acetamido-4′isothiocyanostilbene-2, 2′-disulphonic acid (SITS) strongly inhibited the oocytes anion currents and trophozoites growth. Experiments at diverse pHs suggested that EhClC-A is not a Cl/H+ exchanger, but it is an ion channel that could be involved in pH regulation. EhClC-A may also participate in cell volume regulation. As far as we know, EhClC-A is the first chloride channel characterized in protozoa.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Entamoeba histolytica is the enteric protozoan responsible of human amoebiasis, a disease that causes up to 100 000 deaths each year worldwide (Walsh, 1986). The pathogenic mechanism of this parasite has three main events: trophozoite-adherence, contact-dependent cytolysis and phagocytosis (Orozco et al., 1982). In these events participate adhesins, cysteine proteinases, pore-forming proteins, acid phosphatases, cytoskeleton proteins, small GTPases and proteins involved in signal transduction (Laughlin and Temesvari, 2005). Ion transport through plasma membrane seems to play also a crucial role in E. histolytica pathogenesis. The flux of calcium ions induced by the ionophore A23187 stimulates the release of pore-forming polypeptides, called amoebapores (Young et al., 1982), that are inserted on the target cell membranes after trophozoite adherence (Leippe, 1997). This ionophore also induces exocytosis of acidic vesicles (Ravdin et al., 1988) that probably contain cysteine proteinases, enzymes that participate in target cell lysis (Que and Reed, 2000). In addition, adherence to target cells and cytolysis is inhibited by Na+ and Ca++ channel blockers (Ravdin et al., 1982). The overexpression of EhPgp5, a protein involved in the multidrug resistance event in this parasite (Descoteaux et al., 1992), produced a trophozoite volume increase, and Xenopus laevis oocytes microinjected with the Ehpgp5 cRNA exhibited ion currents that were inhibited by chloride channel blockers (Delgadillo et al., 2002). Thus, Ehpgp5 seems to be involved in cell volume regulation of trophozoites by the activation of chloride currents. However, in spite of the importance of ion transport in the biology and pathogenicity of E. histolytica, there is no information about the molecules involved in ion transport in this parasite.

Ion channels are integral membrane proteins that form pores to allow the passage of specific ions by passive diffusion. Most ion channels undergo conformational changes from closed to open states, and once open, channels allow the passage of thousands of ions. This distinguishes them from transporters and pumps, which also transport ions but only one, or a few at a time. The opening and closing of channels is controlled by various mechanisms, including voltage, the binding of ligands such as intracellular Ca2+ or extracellular neurotransmitters and post-translational modifications of the proteins forming the channels, such as phosphorylation (Jentsch et al., 2004). Anion channels permit the passive diffusion of negatively charged ions (Cl, Br, F, I, NO3) along their electrochemical gradient. They are often named chloride channels, because Cl is the most abundant anion in organisms and hence is the predominant permeating species under most circumstances (Jentsch et al., 2002). The voltage-gated ClC chloride channels perform a wide variety of physiological functions such as the control of electrical excitability (Klocke et al., 1994), the transepithelial transport (Isnard-Bagnis et al., 2003), and pH and cell volume regulation (Jin et al., 2003). The physiological importance of ClC chloride channels is evidenced in human diseases resulting from mutations in these proteins (Koch et al., 1992; Steinmeyer et al., 1994; Lloyd et al., 1996; Simon et al., 1997). Nine different members of ClC channels have been identified in mammals (Jentsch and Gunther, 1997), and ClC genes have been found in animals (Jentsch et al., 1990; Adachi et al., 1994; Miyazaki et al., 1999; Bianchi et al., 2001; Catalan et al., 2002), plants (Hechenberger et al., 1996; Lurin et al., 2000), yeast (Greene et al., 1993; Zhu and Williamson, 2003), archaebacteria (Jentsch et al., 1999) and eubacteria (Jentsch et al., 1999; Purdy and Wiener, 2000). However, there are not reports on the characterization of anion channels in protozoa.

Here, we present the identification of two E. histolytica genes (Ehclc-A and Ehclc-B) encoding for polypeptides with 25–30% identity to ClC channels of different organisms. We also performed the functional characterization of the EhClC-A channel. Structural and physiological results suggest that EhClC-A is related to the mammalian ClC-2 subfamily.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Identification and in silico characterization of two E. histolytica genes encoding putative ClC channels

The voltage-dependent chloride channels (ClC) are involved in functions common to all cells and they are widely distributed in organisms (Jentsch et al., 1999). The putative role of these proteins in E. histolytica biology, including pathogenicity, prompted us to search for their presence in this parasite and study their functions.

By blast searching of the E. histolytica genome project database (http://www.tigr.org/tdb/e2k1/eha1/) using the consensus motifs of ClC channels that conform their selectivity filter (Dutzler, 2004) we identified two genomic sequences. The 18.t00013 genomic sequence, named here Ehclc-A, contains 2069 bp with a putative 62 bp intron. It encodes a 668 amino acids (aa) polypeptide with a predicted molecular weight of 74.5 kDa (EhClC-A) that includes amino acid sequences identical or similar to those used for the search at positions 150–155 (GSGVPE), 192–197 (GKEGPF) and 245–250 (GGLLFS) (Fig. S1). The other genomic sequence (12.t00056), named Ehclc-B, has 2168 bp with two 77 and 81 bp putative introns. The predicted cDNA presents an open reading frame of 2010 bp encoding for a 669 aa polypeptide with a molecular weight of 74 kDa (EhClC-B). EhClC-B contains the sequences used for the search at positions 151–156 (GSGVPE), 193–198 (GKVGPS) and 246–251 (GGLLFS) (Fig. S1). Interestingly, EhClC-B has a valine residue at position 195 instead of the conserved glutamate residue. This change has been described as responsible of abolishing the voltage-dependent gating of ClC channels (Fahlke et al., 1997; Dutzler et al., 2003; Estevez et al., 2003; Niemeyer et al., 2003; Dutzler, 2004), suggesting a lack of voltage-dependent gating in EhClC-B. Reverse transcription polymerase chain reaction (RT-PCR) assays using specific primers from each ClC gene showed the amplification of the expected cDNA bands of 303 and 1917 bp corresponding to the Ehclc-A and Ehclc-B transcripts respectively (Fig. 1A). These results indicate that both ClC genes are expressed in E. histolytica trophozoites.

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Figure 1. Expression of E. histolytica ClCs and their phylogenetic relationship with mammalian ClCs. A. RT-PCR of Ehclc-A and Ehclc- B. E. histolytica RNA was reverse transcribed using superscript II and an oligo-dT primer. Then, PCR assays were performed using the cDNA and specific primers for each ClC gene. Lane 1, molecular size markers. Lane 2, PCR control using primers for Ehclc-A and RNA as template (RT enzyme was omitted). Lane 3, RT-PCR using primers for Ehclc-A. Lane 4, RT-PCR using primers for Ehclc-B. B. Phylogenetic relationship of E. histolytica ClCs with other ClCs. The amino acid sequences of E. histolytica ClC channels were compared with those of the nine ClC proteins of human (Accession numbers: ClC-1, P35523; ClC-2, P51788; ClC-3, P51790; ClC-4, P51793; ClC-5, P51795; ClC-6, P51797; ClC-7, P51798; ClC-K1, P51800; ClC K2, P51801), ClCs of Saccharomyces cerevisiae (GIF1; Accession number NP012574), E. coli (Accession number P37019), S. typhimurium (Accession number NP459208) and putative ClCs of Leishmania major (Accession numbers NP_047043 and CAC22644), Trypanosoma brucei (Accession numbers EAN76853 and EAN80614), Trypanosoma cruzi (Accession numbers XP_814380 and XP_810521) and Trichomonas vaginalis (locus 93905.m00163 and 81403.m00052 in http://www.tigr.org/tdb/e2k1/tvg/). Data were submitted to phylogenetic analysis by the neighbour-joining method. Numbers of the nodes represent the bootstrap proportions. E. histolytica ClCs are indicated in bold. The scale bar indicates 0.2 substitutions at each amino acid position.

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EhClC-A and EhClC-B exhibited 58% identity between them and 25–30% identity with several ClC channels of different organisms. Like these proteins, both E. histolytica ClCs contain 12 hydrophobic regions (D1-D12) and two cystathionine-beta-synthase (CBS) domains in their cytoplasmic carboxy-terminus (Fig. S1). In other organisms, it has been demonstrated that CBS domains participate in intracellular trafficking (Carr et al., 2003), and they also have a critical role in the ClC-1 channel function (Hebeisen et al., 2004). In addition, EhClC-A and EhClC-B contain a potential N-glycosylation site into the putative extracellular component between D8 and D9 domains, which is highly conserved in the ClC family (Fig. S1).

Entamoeba dispar, a non-pathogenic amoeba morphologically undistinguishable of the pathogenic E. histolytica (Diamond and Clark, 1993), is an interesting tool for comparative studies on identified E. histolytica proteins. Therefore, we search for orthologous genes of Ehclc-A and Ehclc-B in the currently in progress E. dispar genome project (http://www.sanger.ac.uk/Projects/E_dispar). This analysis revealed two sequences encoding polypeptides with 98.4% and 100% identity to positions 75–220 and 467–669 of EhClC-B, respectively, whereas other two genomic sequences encode polypeptides with 96.4% and 93.4% identity to positions 44–225 and 385–660 of EhClC-A respectively (data not shown).

To determine the homology of EhClC-A and EhClC-B with different ClC subfamilies, we aligned the predicted E. histolytica ClCs with the nine human ClCs and with ClCs of Saccharomyces cerivisiae, Escherichia coli and Salmonella typhimurium. We also included in this alignment putative ClCs encoded by nucleotide sequences of Leishmania major, Trypanosoma cruzi, Trypanosoma brucei and Trichomonas vaginalis deposited in the GenBank or in The Institute for Genomic Research databases (http://www.tigr.org). Based on this alignment we constructed a phylogenetic tree. In concordance with previous studies (Jentsch et al., 1999), this tree showed that: (i) mammalian ClCs are distributed into three subfamilies; (ii) the yeast ClC is situated in the branch comprising mammalian ClC-3, ClC-4 and ClC-5; and (iii) bacterial ClCs appeared in a branch distinct to the eukaryotic ClCs (Fig. 1B). Our tree also showed that both E. histolytica ClC channels belong to the branch that includes mammalian ClC-1, CLC-2, ClC-K1 and ClC-K2 (Fig. 1B). It has been reported that these proteins function as plasma membrane ClC channels that participate in pH and cell volume regulation (Nilius and Droogmans, 2003). In contrast, the putative ClCs of L. major, T. brucei and T. cruzi were grouped in the other two eukaryotic branches that include mammalian ClCs located in internal membranes (Fig. 1B) (Jentsch et al., 1999). Interestingly, the two putative ClCs of T. vaginalis were situated in the branch comprising the prokaryotic ClCs (Fig. 1B), suggesting that they are the most ancient eukaryotic ClCs identified until now.

Cloning of the Ehclc-A cDNA

To initiate the physiological characterization of the E. histolytica ClC channels we studied the EhClC-A protein, because according to its structural characteristics, theoretically, it could exhibit a voltage-dependent gating and it should be a plasma membrane protein. First, we obtained its cDNA from an E. histolytica cDNA library using as a probe a 303 bp PCR DNA fragment generated with primers designed from encoding regions of the GSGVPE and GGLLFS motifs of the Ehclc-A gene and total E. histolytica DNA as template. We obtained a 2061 bp cDNA containing the complete open reading frame of Ehclc-A and 5′- and 3′-untranslated regions of 18 and 39 bp respectively. Comparison between cDNA and genomic sequences confirmed the presence of the predicted intron found in the Ehclc-A gene and sequences of the two exons were identical to the cDNA sequence (data not shown). The Ehclc-A cDNA clone was used to produce the transcript for physiological experiments.

Microinjection of the Ehclc-A cRNA in Xenopus oocytes

To express and characterize the EhClC-A channel, we produced and injected its cRNA in X. laevis oocytes and then, ionic currents were measured by two-electrode voltage clamping. Two days after the cRNA injection, inwardly rectifying currents (i.e. outward flow of ions) at negative potentials were recorded (Fig. 2A). Amplitude of these currents were higher 6 days after injection (Fig. 2A), indicating a correlation between time after injection and current amplitude, as it occurs for the expression of electrogenic molecules in Xenopus oocytes (Tzounopoulos et al., 1995). Six days after cRNA injection, stimulation at −160 mV elicited currents of −0.33 ± 0.01 µA that were approximately six times larger that those produced in oocytes injected with water (P < 0.0001) (Fig. 2A and B).

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Figure 2. Ion currents of Xenopus oocytes injected with the Ehclc-A cRNA. A. Representative membrane currents recorded from oocytes 2 and 6 days after injection with Ehclc-A cRNA, and 6 days after injection with distilled water. Membrane was held at −30 mV, steps pulses for 5 s to various potentials between −160 and +20 mV in 20 mV intervals were applied and recorded, and currents were superimposed. B. I–V curves for membrane currents of oocytes injected with Ehclc-A cRNA and those injected with distilled water. Bars indicate standard deviation. n, number of measurements in one oocyte. N, number of injected oocytes. *P < 0.0001 between oocytes injected with Ehclc cRNA and those injected with water.

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It has been reported that injection of oocytes with a high concentration of cRNA (≥ 100 ng µl−1) may result in the induction of endogenous calcium-activated chloride currents (Attali et al., 1993). To avoid these endogenous currents, oocytes used in our experiments were incubated in solution containing low calcium concentration and they were injected only with 25 ng of cRNA (0.5 ng µl−1). In addition, the control oocytes injected with 25 ng of a non-related cRNA (elongation factor 1 α of Xenopus) showed only small currents similar to those displayed by oocytes injected with distilled water (data not shown). Therefore, the Ehclc-A cRNA generated the inward currents in injected oocytes. Like currents produced by mammalian ClC-2 channels, EhClC-A induced currents that were activated rapidly upon hyperpolarization (Fig. 2A). These results suggested that EhClC-A is a chloride ion channel, probably related to ClC-2 subfamily.

Electrophysiological properties of EhClC-A

The permeability of different anions through EhClC-A was determined by replacing in the register solution the 48 mM Cl with equimolar Br, I, F and NO3 and analysing the ion currents displayed by cRNA-injected oocytes (Fig. 3A). The reversal potential (Erev) values of these currents were −26.44 ± 0.98 mV in Cl solution (n = 5, N = 8), −19.07 ± 1.50 mV in Br solution (n = 5, N = 8), −11.17 ± 1.35 mV in F solution (n = 5, N = 8), −6.67 ± 0.95 mV in I solution (n = 5, N = 8) and +4.33 ± 1.65 mV in NO3 solution (n = 5, N = 8) (Fig. 3B). Thus, the sequence of anion permeability was Cl > Br > I > F >> NO3 with respect to their ability to stimulate inward currents. This anion selectivity is similar to that showed by mammalian ClC-2 (Fahlke, 2001), strengthening the idea that EhClC-A may be the orthologous protein of mammalian ClC-2.

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Figure 3. Permeability of anions through Ehclc-A channels. A. I–V curves for membrane currents of oocytes injected with Ehclc-A cRNA and in the presence of external solutions with Cl, Br, I, F and NO3. Stimulation protocol used was as in Fig. 2, but only values registered up to −60 mV are presented here. 48 mM extracellular Cl was replaced with equimolar concentration of the indicated anions during the entire pulse protocol. B. Erev values for currents elicited in the presence of different anions. Bars indicate standard deviation. n, number of measurements in one oocyte; N, number of injected oocytes.

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The sensitivity of EhClC-A currents to Cl channel blockers was determined using DIDS (1 mM), SITS (1 mM), 9-AC (1 mM), DPC (1 mM) and niflumic acid (200 µM). Currents of cRNA-injected oocytes in the absence of any blocker were recorded, then, oocytes were sequentially perfused with the candidate inhibitors and currents were registered again. Peak currents obtained from oocytes stimulated at −160 mV after drug application were normalized with respect to those produced in the absence of drug (n = 5, N = 8). DIDS, 9-AC, DPC and niflumic acid had no significant effect on currents displayed by injected oocytes (data not shown). In contrast, 1 mM SITS decreased in more than 60% the peak currents of the injected oocytes (Fig. 4A). Next, we analysed the dose–response inhibition of SITS on the oocytes currents. Again we normalized the peak currents of oocytes stimulated at −160 mV after drug treatment with respect to currents elicited in the absence of the drug. Currents produced by oocytes in the presence of 0.6 mM SITS were similar to those produced in the absence of drug, whereas 0.8 and 1 mM of SITS reduced the currents in 22% and 62% respectively (P < 0.001) (Fig. 4A).

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Figure 4. Effects of SITS on currents displayed by EhClC-A and on trophozoites growth. A. Effect of SITS on oocytes currents. Currents elicited by oocytes after 6 days injection with the Ehclc-A cRNA were recorded. Then, oocytes were perfused with 0.6, 0.8 and 1 mM of SITS and currents were registered again. Stimulation protocol used was as in Fig. 2. Peak currents obtained in the presence of the channel blocker at −160 mV were normalized to those obtained in the absence of the drug (n = 5; N = 8). Bars indicate standard deviation. *P < 0.0001 for current amplitude between the absence and the presence of SITS. B. Effect of SITS on trophozoites growth. E. histolytica trophozoites were grown in the absence of drugs or in the presence of 0.6, 0.8 and 1 mM SITS. Then, the number of cells and their viability was registered each 12 h. Bars indicate standard deviation.

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Effect of Cl channel blockers on the growth of E. histolytica trophozoites in culture

To test the importance of Cl channels in the viability of E. histolytica we evaluated the effect of Cl channel blockers on the growth of trophozoites in the culture medium. We also examined the dose–response inhibition of SITS on the trophozoites growth. These assays showed that 0.6, 0.8 and 1 mM of SITS inhibited the trophozoites growth in 14%, 28% and 89% with respect to the trophozoites grown in the absence of drugs (Fig. 4B) respectively. Similar to the effect on the currents displayed by injected oocytes, DIDS, 9-AC, DPC or niflumic acid had not effect on the trophozoites growth (data not shown). These results suggest that EhClC-A could be essential in the physiology of E. histolytica trophozoites.

Effect of changes in pH on the currents elicited by EhClC-A

Recent studies have been shown that the ClC of E. coli and mammalian ClC-4 and ClC-5 are not ion channels, but rather they are Cl/H+ exchange transporters (Accardi and Miller, 2004; Picollo and Pusch, 2005; Scheel et al., 2005). To determine whether EhClC-A is an ion channel or it is a transporter we analysed the currents displayed by oocytes injected with the Ehclc-A cRNA at different pHs (Fig. 5A). In these experiments the Erev values of currents displayed by oocytes perfused with register solutions at pH 7.6, 6.6, 5.6 and 4.6 were almost identical (Fig. 5B). These results indicated that there was not H+ transport through EhClC-A. In addition, the recordings of currents displayed by oocytes perfused with register solutions containing different Cl concentrations showed the predictable displacement of the current-voltage curves (Fig. 5C). The Erev values obtained using external solutions with 48 and 96 mM of NaCl (−23 and −40.1 mV, respectively) are closely to the Nernst equilibrium potential expected for perfect Cl selectivity (−22 and −39.5 mV). These results strongly suggest that EhClC-A is not a transporter, but it is a chloride ion channel. These results were expected by the homology of EhClC-A to mammalian ClC-2, which are clearly associated with ion channel activity (Picollo and Pusch, 2005).

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Figure 5. Effect of pH and Cl concentration in external solution on currents produced by EhClC- A. A. I–V curves for membrane currents of oocytes injected with Ehclc-A cRNA when the pH of external solution was changed from pH 7.6 to different pHs. *P > 0.0001 with respect to the control at pH 7.6. B. Erev values obtained from the I–V curves under different pHs. C. I–V currents of oocytes injected with Ehclc-A cRNA using 96 and 48 mM of NaCl in the extracellular solution. Stimulation protocol was as in Fig. 2. Bars indicate standard deviation. n, number of measurements in one oocyte; N, number of injected oocytes.

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Interestingly, after 1 min perfusion with solution at pH 5.6, currents exhibited by injected oocytes had a significant increase (Fig. 5A). When they were stimulated at −160 mV the current amplitude augmented up to −0.47 ± 0.01 µA (42% more with respect to the currents elicited at pH 7.6). In contrast, oocytes injected with distilled water did not modify their endogenous currents after perfusion with solution at pH 5.6 (data not shown). These results suggest that the EhClC-A channel could be involved in pH regulation in E. histolytica.

Effect of hypotonicity on the currents displayed by EhClC-A

To test whether EhClC-A, like mammalian ClC-2, could also be involved in cell volume regulation, oocytes injected with Ehclc-A cRNA were placed in a hypotonic register solution to analyse the currents displayed by them after voltage stimulation. Quickly (1 min), hypotonicity increased the currents displayed by these oocytes. When they were stimulated at −160 mV, currents augmented up to −0.65 ± 0.03 µA, 97% more with respect to the values obtained at isotonic condition (Fig. 6). In addition, reperfusion with isotonic solution (wash out) partially reversed current amplitude (Fig. 6). Oocytes injected with distilled water perfused with hypotonic solution and reperfused with isotonic solution did not modify their endogenous currents (data not shown). These results suggest that, as mammalian ClC-2 channels, EhClC-A may be also implicated in cell volume regulation.

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Figure 6. Effect of external hypotonicity on EhClC-A currents. I–V curves for membrane currents of oocytes injected with Ehclc-A cRNA when external solution was changed from isotonic solution to hypotonic solution and changed back to isotonic solution (wash out). Stimulation protocol was as in Fig. 2. Bars indicate standard deviation. n, number of measurements in one oocyte; N, number of oocytes. *P < 0.0001 with respect to the control at isotonic condition.

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Subcellular localization of EhClC-A in E. histolytica trophozoites

To determine the subcellular localization of EhClC-A in the trophozoites, we obtained specific antibodies against EhClC-A (anti-EhClC-A) using a synthetic polypeptide situated in the N-terminal region of EhClC-A that is absent in EhClC-B (Fig. S1). In Western blot experiments employing total extracts of trophozoites, anti-EhClC-A antibodies detected two 75 and 80 kDa proteins (Fig. 7A, lane 1). The lower band recognized by anti-EhClC-A corresponds to the molecular weight expected from the gene sequence. The upper band could be the EhClC-A polypeptide with post-translational modifications, as N-glycosylation. The preimmune serum, used as a control, did not detect any band (Fig. 7A, lane 2). Immunofluorescence assays using the anti-EhClC-A antibodies demonstrated that EhClC-A is situated mainly at the plasma membrane of the trophozoites, although fluorescent spots were also found in the cytoplasm (Fig. 7B). No signal was seen in trophozoites incubated with the preimmune serum or when the anti-EhClC-A antibodies were omitted (data not shown). These results confirm that EhClC-A is a plasma membrane protein.

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Figure 7. Immunolocation of EhClC-A in E. histolytica trophozoites. A. Western blot on total extracts of E. histolytica trophozoites using anti-EhClC-A antibodies (lane 1), or preimmune serum (lane 2). Arrows show the 75 and 80 kDa polypeptides recognized by antibodies against EhClC-A. B. Immunolocalization of EhClC-A. E. histolytica trophozoites were fixed, permeabilized and incubated with rabbit anti-EhClC-A antibodies, and then, with a secondary anti-rabbit-fluoresceinated antibody. Then, cells were observed through confocal microscopy. (a) Image of an optical section corresponding to the middle of trophozoites. (b) Phase contrast.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Conservation of ClC chloride channels in widely divergent organisms confirms their universal and fundamental physiological roles. Until now, the presence of ClCs had not been described in protozoa, although several reports have suggested their existence and their role in these organisms. Desai et al. (2000) identified unusual chloride currents on the membrane of red blood cells (RBCs) infected with Plasmodium falciparum. Since then, controversy has arisen about whether these currents are due to channels encoded by the parasite or they result from an endogenous host channel modified. Whole-cell patch-clamp experiments using infected RBCs revealed currents that were absent when parasites were cultured in RBCs from cystic fibrosis donors with the homozygous F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) chloride channel (Verloo et al., 2004), suggesting that the parasite activates an endogenous chloride channel tightly linked to CFTR. However, electrophysiological characterization of cells infected with two distinct Plasmodium isolates suggested that an anion channel is encoded by the parasite (Alkhalil et al., 2004), but this putative channel has not been identified. On the other hand, genes encoding putative ClC chloride ion channels of L. major, T. brucei, T. cruzi and T. vaginalis were deposited in databases, but we did not find any report on their characterization. Thus, EhClC-A and EhClC-B seems to be the first chloride channels characterized in protozoa.

Comparison of EhClC-A and EhClC-B with the mammalian ClCs revealed that both E. histolytica channels are members of the eukaryotic branch comprising ClC channels located in the plasma membrane of the cells. Immunofluorescence assays in trophozoites using antibodies against EhClC-A confirmed that it is located in the plasma membrane. In contrast, the ClCs of yeast, L. major, T. brucei and T. cruzi are more related to mammalian channels located in internal membranes, suggesting a similar location for these proteins in these organisms. According with our alignment, the two putative ClCs of T. vaginalis are more related to prokaryotic than to eukaryotic ClCs. Recent functional studies on the E. coli ClC indicate that it is a Cl/H+ exchanger (also named antiporter) (Accardi and Miller, 2004; Picollo and Pusch, 2005; Scheel et al., 2005). Due to the higher similarity of T. vaginalis ClCs to the E. coli ClC, we suggest that the T. vaginalis ClCs could function also as Cl/H+ exchangers. In contrast, the physiological properties of EhClC-A indicated that it is an ion channel. However, a more precise method to determine the possible H+ flux through EhClC-A, as the pH-sensitive microelectrode used by others (Picollo and Pusch, 2005), is needed to discard that EhClC-A could be an antiporter.

In most ClCs a conserved glutamate residue of the G(K/R)EGP motif occupies one of the chloride binding sites in the close conformation of the channel, occluding the extracellular side of the transport pathway (Dutzler, 2004). In the E. coli ClC, the mutation of this glutamate to a neutral residue produces that the channel acquire a constitutive open conformation (Dutzler, 2004). Similar mutation abolishes voltage-dependent gating of the Torpedo ray ClC-0, and mammalian ClC-1 and ClC-2 channels (Fahlke et al., 1997; Estevez et al., 2003; Niemeyer et al., 2003; Dutzler, 2004). In the E. coli ClC and in mammalian ClC-4 and ClC-5 antiporters, the mutation of the conserved glutamate residue also abolish the H+ transport, but not the Cl passage (Accardi and Miller, 2004; Picollo and Pusch, 2005; Scheel et al., 2005). EhClC-B, like mammalian ClC-K1 and ClC-K2, has a valine residue instead of the conserved glutamate in this motif (Fig. S1), suggesting that in addition to a lack of voltage-dependent gating, this E. histolytica protein may transport Cl, but not H+. Thus, EhClC-A and EhClC-B could be considered as the most ancient members identified as plasma membrane chloride channels in eukaryotes.

In mammals, a small protein named Barttin functions as the β-subunit of ClC-K members, necessary for the functional expression of these channels (Estevez et al., 2001). As mentioned before, EhClC-B could be functionally related to mammalian ClC-K members, and the existence of a β-subunit for EhClC-B with similar function to the Barttin protein remains to be determined.

Xenopus oocytes injected with Ehclc-A cRNA elicited currents with similar electrophysiological features to ClC-2 subfamily, in terms of I–V relationships and ion selectivity (Jentsch et al., 1999), suggesting that EhClC-A is more related to ClC-2 channels, which may be involved in transepithelial salt and water transport, in pH adjustment and cell volume regulation (Waldegger and Jentsch, 2000). The EhClC-A currents were activated at pH 5.6 and in hypotonic solution, suggesting that EhClC-A could be implicated in pH and volume regulation.

The Cl channel blocker SITS inhibited in similar dose-dependent way both, the currents displayed by injected oocytes and the trophozoites growth, suggesting that EhClC-A may perhaps has an essential function in E. histolytica physiology. To confirm this, we attempted to obtain parasite populations depleted of EhClC-A by antisense RNA technology. However, after several efforts we did not obtain viable trophozoites following transfection with the antisense construction, whereas transfectants with the EhrabB gene (Rodriguez et al., 2000), used as control, were easily obtained (data not shown). These results support the idea that EhClC-A has an important role in the trophozoites viability, making this protein a good target for the search of specific drugs against the parasite. However, it would be useful to confirm the role of EhClC-A in the trophozoites viability determining the effect of the blockage of its expression by antisense approach using a tetracycline regulatable vector (Sahoo et al., 2003; 2004) or using antibodies that obstruct the ion transport through EhClC-A.

Xenopus oocytes express different endogenous chloride channels in their plasma membrane, including hyperpolarization-activated chloride channels (Kowdley et al., 1994). However, the currents exhibited by these endogenous channels clearly differ from EhClC-A currents, because endogenous currents are insensitive to external acidification (Kowdley et al., 1994), which activates EhClC-A. Most importantly, EhClC-A has a Cl > I selectivity, while endogenous channels conduct iodide better than chloride (Kowdley et al., 1994). Oocytes also express endogenous swelling-activated chloride currents, which depend on the follicular cell layer enclosing the oocyte (Arellano and Miledi, 1995). These currents decay within 2 days after defolliculation (Arellano and Miledi, 1995). Hence, they were absent in the oocytes used in this work. On the other hand, currents elicited by oocytes injected with distilled water were not affected by changes in pH or by hipotonicity. Therefore, the contribution of endogenous Cl currents in our experiments is expected to be negligible.

In conclusion, we identified here two ClC chloride channels in E. histolytica. By its cellular localization and electrophysiological properties, EhClC-A could be considered the most evolutionarily distant member identified for the ClC-2 subfamily. Meanwhile, due to its structural features EhClC-B could be considered as an ancestor of the ClC-K group. EhClC-A could be a suitable target for novel drugs against amoebiasis.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Entamoeba histolytica cultures

Trophozoites of clone A, strain HM1:IMSS (Orozco et al., 1983), were axenically cultured in TYI-S-33 medium and harvested during the logarithmic growth phase (Diamond et al., 1978). To test the effect of Cl blockers on trophozoites, 1.6 × 105 cells were incubated at 37°C in 0.5 ml of TYI-S-33 medium in the absence or in the presence of any the following Cl channel blockers: 4-4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS), 4-acetamido-4′isothiocyanostilbene-2,2′-disulphonic acid (SITS), anthracene-9-carboxylic acid (9-AC), diphenylamine-2-carboxylic acid (DPC) or niflumic acid. All drugs were prepared as stock solutions in dimethyl sulphoxide (DMSO) and used at the concentrations described in the text. Then, the number of trophozoites and their viability, measured by trypane blue exclusion, was registered each 12 h.

Identification and in silico characterization of E. histolytica genes encoding for ClC chloride channels

To identify putative ClC encoding genes in E. histolytica, we performed a blast homology search on the E. histolytica genome project database (http://www.tigr.org/tdb/e2k1/eha1/) (the sequencing effort of The Institute for Genomic Research is part of the International Entamoeba Genome Sequencing Project, and is supported by award from the National Institute of Allergy and Infectious Diseases, National Institutes of Health), using as probes the GSGIPE, GKEGPF and GGLVFS amino acids sequences, which are conserved motifs in the ClC family that form their selectivity filter (Dutzler, 2004).

The predicted ClC channels of E. histolytica were characterized  in silico using the software deposited in the Expasy Proteomic Service (http://www.expasy.org) and the NCBI Home Page (http://www.ncbi.nlm.nih.gov). Phylogenetic analysis were conducted using MEGA version 3.0 (Kumar et al., 2004).

Reverse transcription PCR assays

Total E. histolytica RNA was obtained using the TRIZOL reagent (Gibco BRL) according to the manufacturer recommendations. cDNA was synthesized using an oligo-dT primer and the superscript II reverse transcriptase (Invitrogene) and then, PCR amplifications were carried out using primers corresponding to the Ehclc-A (sense: 5′-GGATCAGGTGTA CCAGAA-3′; antisense: 5′-TGAAAATAAAAGACCACC-3′) and Ehclc-B (sense: 5′-ATGGCAAAGATTAAAAAAAT-3′; antisense: 5′-TTAGAACAAAATTTTCATTTG-3′) genes.

Cloning of the cDNA encoding the EhClC-A channel

Total  E. histolytica DNA was obtained as described (Sanchez et al., 1994). Next, PCR assays were performed using primers corresponding to the GSGVPE (sense: 5′-GGATCAGGTGTACCAGAA-3′) and GGLLFS (antisense: 5′-TGAAAATAAAAGACCACC-3′) motifs encoded by the 18.t00013 sequence (http://www.tigr.org/tdb/e2k1/eha1/), which was named Ehclc-A. Then, the 303 bp product of the PCR was random primer labelled with [α-32P]-dATP (Amersham) and used as a probe on a cDNA library of E. histolytica cloned in the λZapII vector (Sanchez et al., 1994). Positive clones were in vivo exscinded to obtain the cDNA in the pBluescript SK+ vector (pBS). Clones were fully sequenced on both strands using automatic sequencing. The sequence data have been submitted to the DDBJ/EMBL/GeneBank databases under Accession number AJ554044.

Production of the Ehclc-A cRNA

To characterize the electrophysiological properties of EhClC-A, the pBS plasmid containing its cDNA was digested with KpnI enzyme. The Ehclc-A cRNA was in vitro synthesized through the T3 promoter and a 5′-cap was added using the mMessage mMachine transcription kit (Ambion). After incubation with DNase, the cRNA was extracted with phenol-chloroform. Its integrity was analysed through denaturing agarose-gel electrophoresis and it was quantified using UV spectrometry.

Preparation of X. laevis oocytes

Bags of the ovary of African clawed frog X. laevis (anaesthetized with ice and 0.1% 3-aminobenzoic acid ethyl ester) were surgically removed and placed in ND48 solution (NaCl 48 mM, KCl 2 mM, CaCl2 1.8 mM, MgCl2 1 mM, HEPES 5 mM; pH 7.6) supplemented with 100 µg ml−1 of gentamicin. Oocytes were either mechanically singled out, using a platinum loop, or obtained by treatment with Collagenase IA (Sigma) 2 mg ml−1, during 90 min to remove their follicular envelopes. Oocytes were stored at 16°C up to 7 days before using in modified Barth's solution (NaCl 88 mM, KCl 1 mM, Ca(NO3)2 0.33 mM, CaCl2 0.41 mM, MgSO4 0.82 mM, NaHCO3 2.4 mM, HEPES 15 mM; pH 7.6), supplemented with 100 µg ml−1 of gentamicin.

Electrophysiological analysis

Injection pipets with tips from 0.5 to 1 µm in diameter were baked at > 150°C to destroy RNases and were mounted in a Drummont Nanoject autoinjector (Drummond). Ehclc-A cRNA 25 ng per 50 nl of H2O was injected into the oocytes under microscopic management. As controls, we used oocytes injected with DEPC-treated H2O. The injected oocytes were incubated at 16°C for 2–6 days in modified Barth's solution for translation, processing and embedding of the mature proteins into the cell membrane. Oocytes were examined by two electrode voltage clamping using a Dagan amplifier and pClamp 6.0 software (Axon Instruments). Current-injecting and potential-measuring electrodes were filled with 3 M KCl and had resistances of 0.5–1.5 and 1.5–2.5 MΩ respectively. The bath solution was connected to the ground via a low-resistance bridge containing 2% agarose in 3 M KCl. Oocytes were voltage clamped at a holding potential of −30 mV and 5 s voltage steps were applied from −160 to +20 mV in 20 mV increments. Recordings were performed in ND48 solution at room temperature. To determine the ion permeability, currents displayed by injected oocytes were registered in ND48 register solution. Then, the 48 mM NaCl was sequentially replaced with equimolar amounts of NaI, NaBr, NaF or NaNO3. To analyse the effect of Cl channel blockers on the currents displayed by injected oocytes, after register the currents in ND48 solution, oocytes were sequentially perfused with register solution containing DIDS, SITS, 9-AC, DPC or niflumic acid, and then, currents were analysed again. All the Cl blockers were prepared as stock solutions in DMSO and were diluted in the register solution at final concentrations described in the text. The final concentration of DMSO in each solution was ≤ 0.1%, which had no effect on the oocytes membrane currents. For pH modulation experiments, ND48 was buffered to different pHs. Stimulation of oocytes was analysed 1–20 min after replacing solution at pH 7.6 with solutions at different pHs. Hypotonic stimulation was examined 1–20 min after replacing ND48 with the same solution diluted 1:1 in distilled water (ND48-half). Measured values in different oocytes (N) were averaged and they are shown as means ± standard deviation. In graphics, n means number of measurements in the same oocyte. One-way analysis of variance followed by a Student's t-test was used to test for significance.

Antibodies generation and Western blot assays

To obtain specific antibodies against EhClC-A, the peptide QNSFLVHHENKYLKL, located in position 46–60 of this protein (Fig. S1) was synthesized and intradermally inoculated in rabbits as described (Hancock and Evan, 1998). For Western blot assays, total E. histolytica proteins were separated in 10% SDS-PAGE, transferred to nitrocellulose (Towbin et al., 1979) and probed with antibodies against the EhClC-A peptide (1:5000). Then, membranes were incubated with peroxidase-coupled goat anti-rabbit secondary antibodies (1:5000), and reaction was developed with 4-chloro-1-naftol and 0.04% H2O2.

Cellular immunolocalization of EhClC-A

Trophozoites were fixed with 4% paraformaldehyde at 37°C for 40 min and permeabilized with 0.5% Triton X-100 for 30 min at room temperature. Cells were incubated 1 h at 37°C with antibodies against EhClC-A (1:2000), and then, 1 h at 37°C with an anti-rabbit fluorescein-labelled secondary antibody. Cells were observed through a confocal microscope (Leica TCS SP2). Observations were performed in 13 planes from the top to the bottom of each sample, and the distance between scanning planes was 1 µm.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the European Community and CONACyT (México). We thank Norma Vázquez-Díaz, Blanca Estela Reyes-Márquez, Norma Barragán-Andrade and Alfredo Padilla-Barberi for their invaluable technical assistance.

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Alignment of E. histolytica ClC channels with other ClCs.

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