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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Hydroxyl radical activation of a Ca²⁺-sensitive nonselective cation channel involved in epithelial cell necrosis

Instituto de Ciencias Biomédicas and Centro de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile, Santiago 653-0499, Chile

Submitted 22 January 2004 ; accepted in final form 25 May 2004

	ABSTRACT

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In a previous work the involvement of a fenamate-sensitive Ca²⁺-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca²⁺-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe²⁺, exposure of the intracellular side of NSCC to H₂O₂ increased their open probability (P_o) by 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (·OH) scavenger MCI-186 inhibited the effect of H₂O₂, indicating that the increase in P_o was mediated by ·OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to ·OH. The increase in P_o induced by ·OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P_o of both ·OH and DTNB. A similar increase in P_o was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca²⁺. The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis.

Ca²⁺-activated channels; radical oxygen species; oxidative stress

Fenamate-sensitive NSCCs that are activated by intracellular Ca²⁺ and inhibited by intracellular ATP have been identified in several cell types, both native and cultured (5, 9, 12, 15, 21, 22, 33, 42, 45). These channels exhibit single-channel conductances in the range of 15–35 pS, discriminate poorly between Na⁺ and K⁺, and are impermeable to anions and, for the most part, to divalent cations. In addition, NSCC are blocked by the adenine nucleotides ATP, ADP, and AMP on the cytoplasmic side. The physiological role of these channels remains unclear, in part because of the high concentrations of intracellular Ca²⁺ that are generally required for channel activation.

Redox modulation of ion channels is not without precedent. The large-conductance Ca²⁺-sensitive voltage-dependent K⁺ (K_V,Ca) channel from tracheal myocytes is inhibited by oxidizing agents (49). A similar result was observed in reconstituted skeletal muscle K_V,Ca channels incorporated into bilayers (46). In contrast, ryanodine receptor/Ca²⁺ release channel activity is enhanced by endogenous oxidizing molecules (2, 16). Anion channels are also sensitive to redox modulation. The outwardly rectifying chloride channel in bronchial epithelial cells was shown to be irreversibly inhibited by long exposure to ·OH on the cytoplasmic side of the channel (28). Activation of NSCCs by ROS, including superoxide anion, ·OH, and H₂O₂, has been reported in different cell types (5, 24, 27, 31, 33, 36). Furthermore, cells exposed to severe stress conditions exhibit significant ATP depletion and intracellular Ca²⁺ increase, which is paralleled by an increase in ROS. Under these conditions, NSCC would activate and thus participate in the cation flux involved in necrotic cell swelling (5).

In the present study, we have characterized a NSCC of liver-derived epithelial (HTC) cells and examined more closely the mode of action of H₂O₂ on this channel. We found that the H₂O₂ effect on the NSCC channel activity in HTC cells is mediated by the formation of ·OH, which most probably targets residues located at the intracellular aspect of the NSCC channel. We also found that the effects of H₂O₂ are mimicked by GSSG, a physiological molecule that specifically reacts with sulfhydryl (SH) groups, leading to the conclusion that redox modulation most probably involves a disulfide/thiol exchange of thiol groups of cysteines that may be present in the NSCC protein. In addition, oxidation by GSSG shifts the Ca²⁺ vs. open probability (P_o) curve to the left, indicating that redox modulation of NSCC may play a significant role in signaling mechanisms leading to necrotic cell volume increase and cell demise.

	MATERIALS AND METHODS

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Cell culture and electrophysiological measurements. HTC cells were grown at 37°C in a 5% CO₂-95% air atmosphere in DMEM (GIBCO, Grand Island, NY) supplemented with 5% fetal calf serum (GIBCO), 2 mM L-glutamine, 50 U/ml penicillin-streptomycin, and 2.5 µg/ml amphotericin B (Sigma, St. Louis, MO). For electrophysiological experiments, cells were grown on 22-mm coverslips and directly mounted on the experimental chamber (RC-25, Warner Instruments) installed on the stage of an inverted microscope (IX-70, Olympus America or Nikon Diaphot, Nikon America). Solution changes were done by a gravity-fed perfusion system, and the solution level in the chamber was kept constant by a peristaltic pump. To avoid contamination from anion currents, all experiments were carried out in low-chloride solutions, unless otherwise indicated. The bath (intracellular) high-Ca²⁺ solution contained (in mM) 145 sodium glutamate, 2.6 CaCl₂, 1.3 MgCl₂, 5.6 glucose, and 10 HEPES, pH 7.4, adjusted with Tris. The Ca²⁺-free solution contained (in mM) 145 sodium glutamate, 1.3 MgCl₂, 2 EGTA, 5.6 glucose, and 10 HEPES, pH 7.4, adjusted with Tris. Experiments with oxidizing agents were carried out in a 300 µM free Ca²⁺ solution containing (in mM) 145 sodium glutamate, 1 nitrilotriacetic acid trisodium salt, 0.863 CaCl₂, 1.56 MgCl₂, 5.6 glucose and 10 HEPES, pH 7.4, adjusted with Tris. At this intracellular Ca²⁺ concentration ([Ca²⁺]; [Ca²⁺]_i), small changes in P_o were clearly resolved (see Fig. 2B). The free [Ca²⁺] of solutions used was calculated using the program WinMaxc, version 2.05 (www.stanford.edu/cpatton/maxc.html), with the appropriate Ca²⁺ buffers. The pipette (extracellular) solution, unless indicated otherwise, contained (in mM) 145 sodium glutamate, 2.6 CaCl₂, 1.3 MgCl₂, 5.6 glucose, and 10 HEPES, pH 7.4, adjusted with Tris. Cation selectivity of the NSCC was demonstrated using asymmetrical Na⁺ solutions. In relative permeability experiments for monovalent cations, internal Na⁺ was substituted by equimolar amounts of K⁺, Rb⁺, and Cs⁺. Relative permeability for divalent cations was studied by replacing sodium glutamate in the pipette with 75 mM CaCl₂ or 75 MgCl₂. Changes in liquid junction potential were calculated (6) and voltages were corrected for, when necessary. Patch-clamp pipettes were made from thin borosilicate (hard) glass capillary tubing with an outer diameter of 1.5 or 1.7 mm (Clark Electromedical, Reading, UK), using a BB-CH puller (Mecanex, Geneva, Switzerland). Single-channel currents were recorded with an EPC-7 (List Medical, Darmstadt, Germany) or an Axopatch B (Axon Instruments) amplifier. An agar bridge connected the reference electrode to the bath. Command voltage protocols and single-channel current acquisition were controlled by pCLAMP 8 (Axon Instruments) via a laboratory interface (Digidata 1200 or Digidata 1322A, Axon Instruments). Unless otherwise indicated, membrane holding potential (V_m) was –60 mV. Currents were filtered at 1 KHz with an 8-pole Bessel filter (Frequency Devices) and digitized at 5 kHz. The experiments were performed at room temperature. The number of channels in a given patch, N, was experimentally determined at the beginning of each experiment by exposing the membrane patch to 2.6 mM Ca²⁺ followed by Ca²⁺-free solution (see RESULTS and Fig. 2B). This figure (N) was used to calculate P_o. The number of channels was frequently 6 (range 3–16). When possible, N was also determined at the end of the experiment. In these cases N remained unchanged.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Calcium modulates NSCC activity. A: single-channel activity recorded from a representative inside-out patch containing N = 3 channels exposed to different free [Ca2+]i. From top to bottom: 0, 0.1, 0.3, 1, 2.6, and 10 mM free [Ca2+]i. Dashed lines indicate zero current (C); Ox represents the level of current carried by x channels. B: Ca2+ dependence of open probability (Po). Each membrane patch was exposed to all Ca2+ concentrations (n = 4). Continuous line represents Hill equation fitted to the data with n = 1.54 ± 0.15 and EC50 = 455 ± 33 µM. C: effect of [Ca2+]i on the unitary conductance of NSCC. The number of experiments is shown in parentheses above symbols. Continuous line represents Michaelis-Menten equation fitted to the data with maximum single-channel conductance (max) = 23.6 ± 0.6 pS and Kd = 10.1 ± 0.8 mM. Data are expressed as means ± SE. Error bars are shown if bigger than symbols.

where x stands for open channels from x = 0 to N. In addition, the error associated with the estimation of the total number of channels in the patch was calculated using a minimum square error function of the form

where A_x corresponds to the area of open-state amplitude histograms from x = 1 to N. For all experiments, the minimum error always corresponded to the same value of N determined experimentally.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Hydroxyl radical (·OH) modulates NSCC activity. A: single-channel activity recorded from an inside-out patch containing N = 6 active channels. Control recording was obtained in a sodium glutamate internal solution containing 300 µM free Ca2+ concentration. The intracellular face of the patch was then exposed to 5 mM H2O2 and 5 µM FeSO4, resulting in increased channel activity. After 4 min of washing, 1 mM DTT was applied. At right of each trace, amplitude histograms fitted to a binomial function are shown. These histograms were used for Po calculations as described in MATERIALS AND METHODS. B: Po-vs.-time plot for the tracing shown in A. Vm was kept constant at –60 mV and [Ca2+]i at 300 µM () and in the presence of 5 mM H2O2 plus 5 µM FeSO4 (). Po was measured every 30 s to test for gating stability of the channels. C: summary of results (3–5 patches) showing the effect of H2O2 and FeSO4 and its reversion by DTT. Data are means ± SE. *P < 0.05 with respect to control.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Effect of 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) on NSCC activity. A: single-channel activity recorded from an inside-out patch containing N = 6 channels. Control and experiments were performed as explained in Fig. 4A. At right of each trace, amplitude histograms fitted to a binomial function are shown. These histograms were used for Po calculations as described in MATERIALS AND METHODS. B: Po-vs.-time plot for the tracing shown in A. Vm was kept constant at –60 mV and [Ca2+]i at 300 µM () and in the presence of 0.5 mM DTNB (). Po was measured every 30 s to test for gating stability of the channels. C: summary of results (3–5 patches) showing the effect of DTNB and its reversion by DTT. Data are means ± SE. *P < 0.05 with respect to control.

View larger version (31K): [in this window] [in a new window]   Fig. 7. GSSG and GSH modulate NSCC activity. A: single-channel activity recorded from an inside-out patch containing N = 5 channels. Control and experiments were performed as explained in Fig. 4A. At right of each trace, amplitude histograms fitted to a binomial function are shown. These histograms were used for Po calculations as described in MATERIALS AND METHODS. B: Po-vs.-time plot for the tracing shown in A. Vm was kept constant at –60 mV and [Ca2+]i at 300 µM () and in the presence of 2 mM GSSG (). Po was measured every 30 s to test for gating stability of the channels. C: summary of results (3–7 patches) showing the effect of GSSG and its reversion by GSH. Data are means ± SE. *P < 0.05 with respect to control.

Reagents. All reagents were of analytical grade and were purchased from Sigma and Merck (Darmstadt, Germany). MCI-186 was purchased from Calbiochem (San Diego, CA).

Statistics. Data are presented as means ± SE. Statistical analysis of the data was performed by paired and unpaired Student's t-test and was considered significant at P < 0.05. One-way ANOVA test was performed for multiple treated samples and was considered significant at P < 0.05.

	RESULTS

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Characterization of NSCC in HTC cells. NSCC have been found in different types of liver-derived cell lines and, although they share some functional properties, they differ in their Ca²⁺ permeability (5, 20, 32). In the cell-attached configuration with a pipette solution containing 145 mM sodium glutamate, very few channel openings were observed. After patch excision (inside-out) into a 145 mM sodium glutamate- and Ca²⁺-containing bath solution, channel activity increased significantly, revealing multiple level single-channel currents that were blocked by 100 µM flufenamic acid (not shown; Ref. 45). Experiments showing inward and outward single-channel currents obtained at 2.6 mM [Ca²⁺]_i and symmetrical sodium glutamate (Fig. 1A) were used to construct the current-voltage relationship over the range –80 to 60 mV, depicted in Fig. 1B. The linear fit shows that current reversed close to 0 mV with a slope conductance of 18.9 ± 0.4 pS. Under these experimental conditions, P_o was close to 1 (Fig. 2B) and was voltage independent (not shown). In low (40 mM) internal Na⁺, the current followed the GHK formalism expected for a cation-selective channel (Fig. 1B). To test channel selectivity for different monovalent cations, internal Na⁺ was replaced by equimolar concentrations of K⁺, Rb⁺ and Cs⁺ (Fig. 1, C and D). These replacements did not significantly change unitary current amplitude. The relative permeability (P_Na/P_x) sequence obtained from changes in reversal potential (V_rev) was Cs⁺ (0.87) Na⁺ (1.0) Rb⁺ (1.01) K⁺ (1.11). The permeability for divalent cations was explored next with a pipette solution containing 75 mM CaCl₂ or 75 mM MgCl₂. Under these experimental conditions, no inward currents were detected, therefore suggesting that extracellular divalent cations do not significantly permeate this NSCC. Alternatively, these results might be compatible with divalent cation blockade of the channel. Furthermore, these results confirm that the NSCC is impermeable to anions.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Permeation properties of nonselective cation channels (NSCC). Experiments were carried out in the presence of 2.6 mM intracellular Ca2+ concentration ([Ca2+]i). A: unitary outward and inward currents recorded at the indicated membrane holding potential (Vm) with symmetrical (145 Na+, ) and asymmetrical (40/145, [Na+]i/[Na+]o, ) solutions. Arrows point toward channel closures. B: current-voltage (I-V) relationships of NSCC. Solutions were the same as described above. Slope conductance and reversal potential (Vrev) were 18.9 ± 0.04 pS and –0.5 ± 0.9 mV, respectively, for symmetrical Na+ (, n = 25). In asymmetrical Na+ (, n = 4), the continuous line represents a Goldman-Hodgkin-Katz (GHK) current equation fitted to the data. C: unitary outward currents obtained at Vm = 50 mV for the different monovalent cations (Cl– salt) tested. Arrows point toward channel closures. D: I-V relationships of unitary NSCC currents obtained in 145 mM external sodium glutamate and 145 mM internal XCl, where X stands for the different monovalent cations used: K+ (), Rb+ (), and Cs+ () (n = 10–15). Solid lines represent GHK current equation fitted to the data; permeability ratios are shown in the text. Data are expressed as means ± SE. Error bars are shown if bigger than symbols.

P_o is modulated by internal ATP. Most of the NSCC reported so far are inhibited by intracellular adenine nucleotides. Figure 3A shows representative single-channel current recordings from an excised inside-out membrane patch in symmetrical 145 mM sodium glutamate and 2.6 mM Ca²⁺ in the presence of intracellular ATP. On addition of increasing concentrations of the nucleotide, NSCC activity decreased without changes in unitary conductance. Figure 3B summarizes the effect of internal ATP on the NSCC activity. A Hill function was fitted to the data with n = 0.77 ± 0.08 and IC₅₀ = 32 ± 4 µM. ADP was equally effective in decreasing the activity of NSCC (not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of ATP on NSCC activity. A: single-channel activity recorded from a representative inside-out patch containing N = 3 channels exposed to different intracellular ATP concentrations ([ATP]i) in the presence of 2.6 mM [Ca2+]i. From top to bottom: 0, 50, and 500 µM [ATP]i. B: Hill plot of the effect of ATP. Each membrane patch was exposed to all ATP concentrations (n = 5). Continuous line represents Hill equation fitted to the data with n = 0.77 ± 0.08 and IC50 = 32 ± 4 µM. Data are expressed as means ± SE. Error bars are shown if bigger than symbols.

To rule out the possibility that H₂O₂ could be directly affecting channel activity, we added 5 mM H₂O₂ and 100 µM desferrioxamine to the sodium glutamate, 300 µM Ca²⁺ bath solution. If applied before H₂O₂ addition, desferrioxamine prevents ·OH formation via the Fenton reaction by chelating any traces of heavy metals present in the solution. Under these experimental conditions, P_o values were 0.36 ± 0.02 (control), 0.34 ± 0.02 (desferrioxamine), and 0.35 ± 0.02 (desferrioxamine plus H₂O₂) (n = 6, not statistically different), showing that H₂O₂ and desferrioxamine per se do not modify NSCC activity (Fig. 5A). Moreover, Fe²⁺ by itself (10–100 µM) did not modify P_o (n = 5; P_o control: 0.37 ± 0.04, P_o Fe²⁺: 0.37 ± 0.03). To confirm that ·OH specifically were responsible for the observed increase in P_o, the ·OH scavenger MCI-186 was used. As depicted in Fig. 5B, addition of 0.5 mM MCI-186 to the bath solution prevented the increase in P_o triggered by H₂O₂ plus Fe²⁺ (n = 6). None of the reagents (desferrioxamine, Fe²⁺, H₂O₂, MCI-186, and DTT), alone or combined, affected NSCC unitary conductance.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Desferrioxamine (DFO) and MCI-186 prevent the effect of ·OH on NSCC Po. A: summary of results (n = 6 patches) showing the effect of 100 µM DFO and DFO plus 5 mM H2O2. Data are means ± SE. No statistical difference is observed. B: summary of results (n = 6 patches) depicting the effect of 0.5 mM MCI-186 and MCI-186 plus 5 mM H2O2 and 5 µM FeSO4. Data are means ± SE. No statistically significant difference is observed.

Effect of GSSG on NSCC activity. In physiological conditions, the GSH/GSSG ratio plays a critical role in maintaining intracellular redox balance. Normally, glutathione redox status ([GSH]:[GSSG]) greatly favors GSH. However, during oxidative stress, GSSG accumulates, shifting the redox balance to an oxidizing state (1). To determine whether a physiological oxidizing molecule could modulate NSCC activity, excised inside-out membrane patches were exposed to 2 mM GSSG. Figure 7A depicts a representative experiment before and after addition of GSSG and the respective amplitude histograms fitted to a binomial function. Figure 7B shows the stability plot for the recordings depicted in Fig. 7A. On GSSG addition, P_o increased from 0.34 ± 0.02 to 0.52 ± 0.05 (n = 7, P < 0.05), without changes in unitary conductance. P_o did not return to control values after GSSG removal. However, as shown in Fig. 7, A and C, application of 2 mM GSH as a reducing agent restored P_o to control values (0.38 ± 0.03, n = 3).

Effect of GSSG on NSCC [Ca²⁺]_i-to-P_o relationship. To explore whether the Ca²⁺ sensitivity at more physiological or pathophysiological [Ca²⁺] of the NSCC is affected by oxidation, experiments were performed using the physiological oxidizing agent GSSG. Excised inside-out patches were exposed to 2 mM GSSG and thereafter to different [Ca²⁺]_i. Figure 8 (, continuous line, n = 6–10 for each [Ca²⁺]) depicts the result of such experiments. Compared with nonoxidized channels (dashed line taken from Fig. 2B), oxidized channels show an enhanced sensitivity to Ca²⁺. Channel openings could be consistently observed at low micromolar [Ca²⁺], compared with nontreated channels in which no openings were detected over a 30-min observation period.

View larger version (10K): [in this window] [in a new window]   Fig. 8. Effect of 2 mM GSSG on the [Ca2+]i-Po relationship (). Continuous line represents Hill equation fitted to the data with n = 1.23 ± 0.12 and EC50 = 274 ± 21 µM. Extrapolation to 0 Ca2+ gives a Po of 3%. Dashed line shows the [Ca2+]i-Po relationship under nonoxidized conditions (taken from Fig. 2B).

	DISCUSSION

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In agreement with data collected from several NSCC (12, 22, 47), the Ca²⁺-activated NSCCs in HTC cells were efficiently blocked by adenine nucleotides.

Because of its low oxidizing potential, usually H₂O₂ is not by itself reactive enough with organic molecules (4). Nevertheless, H₂O₂ has the ability to generate highly reactive ·OH through its interaction with redox-active transitional metals (3). ·OH result from the breakdown of H₂O₂ via the Fenton reaction and by interaction of superoxide with H₂O₂ through the Haber-Weiss reaction. Highly reactive ·OH readily reacts with a variety of molecules, such as amino acids and lipids, by removing hydrogen or by addition to unsaturated bonds.

Here we describe that applying H₂O₂ simultaneously with Fe²⁺ to the cytoplasmic face of excised inside-out membrane patches of HTC cells containing Ca²⁺-activated NSCCs produces a significant increase in P_o, without affecting single-channel conductance. Although we cannot exclude the possibility that these conditions may affect other proteins or components associated with the membrane patch, it can be assumed on the basis of previous work in channels in reconstituted systems (34, 46) that the primary target of ·OH is the channel-forming protein. The effect of P_o increase could be ascribed to the generation and oxidizing action of ·OH, as demonstrated by the experiments in which desferrioxamine and MCI-186 were used. The increase in P_o was not reversed by washing, although P_o returned to normal values after the addition of the reducing agent DTT. This observation indicates that ·OH could be targeting some exposed amino acid residues. To address whether the ·OH-induced P_o increase of NSCC could be attributed to the oxidation of free SH residues of cysteine, membrane patches were exposed to DTNB, a hydrophilic agent that reacts specifically with free SH groups in proteins forming disulfide bonds. In the presence of 0.5 mM DTNB, P_o value increased to an extent similar to that observed with H₂O₂ and Fe²⁺, an effect fully reverted by addition of 1 mM DTT. These results indicate that NSCC are modulated by agents that modify the redox condition of SH groups, a result similar to that reported for Ca²⁺-activated K⁺ channels (8, 46). The involvement of cysteinyl residues in the modulation of P_o was investigated further by exposing the NSCC to GSSG. Addition of 2 mM GSSG resulted in a significant increase in P_o to values similar to those observed with H₂O₂-Fe²⁺ or DTNB. The effect of GSSG was completely reversed by its reducing counterpart, GSH, confirming that SH groups of cysteine residues are responsible for the change in the gating behavior of NSCC in HTC cells. Similar results were observed in calf pulmonary artery endothelial cells, in which the activation of a NSCC by tert-butylhydroperoxide was mimicked by GSSG and reversed by GSH (30). Effects of SH modification on channel gating, but not on permeation, have also been reported for cloned K⁺, Na⁺, and Ca²⁺ channels (13, 26, 46). The results presented here are thus compatible with the existence of SH groups susceptible to redox modulation on the cytosolic side of NSCC in HTC cells. In summary, we have extended our previous findings on activation of NSCCs in liver cells by free-radical donors (5) by identifying ·OH as a relevant agent participating in this process. Under pathological conditions associated with oxidative stress, intracellular Ca²⁺ increase, and ATP depletion, NSCC would become activated, generating the influx of cations that results in necrotic cell volume increase. Moreover, the results presented here strongly suggest that ·OH-induced NSCC activation is mediated by specific modifications of cysteine residues, presumably located on the intracellular aspect of the channel protein.

	GRANTS

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This work was supported by Fondo Nacional de Investigación Científica y Tecnológica (Chile) Grant 1010994 and Fondo de Investigación Avanzada en Áreas Prioritarias (Chile) Grant 15010006.

	ACKNOWLEDGMENTS

 
We are grateful to C. Hidalgo for suggestions and critical reading of the manuscript. S. Sala is acknowledged for significant suggestions for the analysis of the data.

Permanent address for Francisco Sala: Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, Campus de San Juan, Apdo. Correos 18, E-03550 Sant Joan d'Alacant, Alicante, Spain.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. Stutzin, Centro de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile, 838-0453, Independencia, Santiago, Chile (E-mail:astutzin{at}bitmed.med.uchile.cl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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