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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

SEA-0400, a potent inhibitor of the Na⁺/Ca²⁺ exchanger, as a tool to study exchanger ionic and metabolic regulation

¹Laboratorio de Biofísica, Instituto de Investigaciones Médicas Mercedes y Martin Ferreira, Córdoba, Argentina; and ²Laboratorio de Permeabilidad Ionica, Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas, Venezuela, and Marine Biological Laboratory, Woods Hole, Massachusetts

Submitted 12 October 2004 ; accepted in final form 13 January 2005

	ABSTRACT

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The effects of a new, potent, and selective inhibitor of the Na⁺/Ca²⁺ exchange, SEA-0400 (SEA), on steady-state outward (forward exchange), inward (reverse exchange), and Ca²⁺/Ca²⁺ transport exchange modes were studied in internally dialyzed squid giant axons from both the extra- and intracellular sides. Inhibition by SEA takes place preferentially from the intracellular side of the membrane. Its inhibition has the following characteristics: it increases synergic intracellular Na⁺ (Na_i⁺) + intracellular H⁺ (H_i⁺) inactivation, is antagonized by ATP and intracellular alkalinization, and is enhanced by intracellular acidification even in the absence of Na⁺. Inhibition by SEA is still present even after 1 h of its removal from the experimental solutions, whereas removal of the cointeracting agents of inhibition, Na_i⁺ and H_i⁺, even in the continuous presence of SEA, releases inhibition, indicating that SEA facilitates the reversible attachment of the natural H_i⁺ and Na_i⁺ synergic inhibitors. On the basis of a recent model of squid Na⁺/Ca²⁺ exchange regulation (DiPolo R and Beaugé L. J Physiol 539: 791–803, 2002), we suggest that SEA acts on the H_i⁺ + Na_i⁺ inactivation process and can interact with the Na⁺-free and Na⁺-bound protonized carrier. Protection by ATP concurs with the antagonism of the nucleotide by H_i⁺ + Na_i⁺ synergic inhibition.

ionic-metabolic interactions

We recently proposed a comprehensive kinetic model for Na⁺/Ca²⁺ exchange regulation in squid axons that takes into consideration intracellular ionic interactions (Na⁺, Ca²⁺, and H⁺) and their connections with two metabolic routes: ATP and phosphoarginine (7). Basically, the model has the following features. First, binding of Ca²⁺ to the intracellular regulatory site is essential for Na_i⁺ or Ca_i²⁺ binding to their transport sites. Second, H_i⁺, but not Na_i⁺, competes with Ca_i²⁺ for the same form of the exchanger, resulting in competitive inhibition of H_i⁺ with Ca_i²⁺ at the regulatory Ca_i²⁺ site. Third, the binding of Na_i⁺ to the protonized carrier allows the binding of a second proton, thus forming a dead end H₂·E₁·Na⁺-inhibitory complex. Fourth, MgATP, through a phosphorylation-requiring process, protects the exchanger by markedly decreasing its apparent affinity for H_i⁺ and Na_i⁺ (6).

Recently, a new, potent, and selective inhibitor of the Na⁺/Ca²⁺ exchanger, SEA-0400 {SEA; 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline}, has been synthesized (19, 25). This compound inhibits the NCX1 isoform with very high affinity (IC₅₀ = 23 nM), seemingly by enhancing Na_i⁺-dependent inactivation (I₁ inactive state in Hilgemann et al.'s notation; see Ref.10) without affecting the Ca_i²⁺-antagonized I₂ inactive state (3, 14, 17). Considering the kinetic model for the exchange regulation described above, it is important to investigate the effects of SEA in the squid nerve. Dialyzed squid giant axons allow accurate control of the intracellular H⁺ concentration ([H⁺]_i), intracellular Na⁺ concentration ([Na⁺]_i), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and intracellular ATP concentration ([ATP]_i), thus making it an excellent preparation in which to explore the site (or sites) of action of SEA. Conversely, SEA can be used as a tool to examine the validity of our kinetic model of ionic and metabolic interactions with the exchanger and to clarify the reaction mechanism of this transporter. In the present article, we provide evidence that 1) SEA does act on the Na_i⁺ inactivation process, but it also interacts with the H·E₁ exchange conformation (6), and 2) the role of ATP protection against H_i⁺ + Na_i⁺ synergic inhibition is supported by the marked reduction of SEA inhibition in the presence of the nucleotide, even at physiological [Na⁺]_i.

	MATERIALS AND METHODS

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Squid giant axons from two species. Loligo pealei (Marine Biological Laboratory, Woods Hole, MA) and Loligo plei (Instituto Venezolano de Investigaciones Científicas, Fundaciencia-Mochima) were euthanized according to procedures approved and applied at MBL and IVIC. They were dialyzed with highly permeable capillaries of regenerated cellulose fibers (OD, 210 µm; ID, 200 µm; molecular mass cutoff, 18 kDa) (Spectrapor no. 132226; Spectrum, Houston, TX). The standard dialysis solution had the following composition (in mM): 385 Tris-MOPS, 45 NaCl, 2 MgCl₂, 285 glycine, and 1–3 Tris-EGTA or BAPTA 3, pH 7.3. Changes in [Na⁺]_i (Cl^– salt) were compensated by isosmolar amounts of MOPS-Tris. Other internal solutions used are described in the figure legends. [Ca²⁺]_i and intracellular Mg²⁺ concentration ([Mg²⁺]_i) were estimated using the WinMaxc computer program (version 2.00, 1999; Chris Patton Hopkins Marine Station, Pacific Grove, CA). ATP (3 mM) was added at 2 mM constant free [Mg²⁺]. The Ca²⁺ pump component of Ca²⁺ efflux and the Na⁺/K⁺ pump were eliminated by adding 100 µM vanadate to the dialysis solutions. The following extracellular solutions were used: 1) dissecting normal artificial seawater containing (in mM) 440 NaCl, 10 CaCl₂, 50 MgCl₂, 10 KCl, and 10 Tris·HCl, pH 7.6; 2) Ca²⁺-free Na⁺ solution seawater [extracellular Na⁺ (Na_o⁺)-0 extracellular Ca²⁺ (0 Ca_o²⁺)] containing (in mM) 440 NaCl, 60 MgCl₂, and 10 Tris-Cl^–, pH 7.6; and 3) Ca²⁺-containing Na⁺ solution seawater (Na_o⁺-Ca_o²⁺) containing (in mM) 440 NaCl, 60 MgCl₂, 3 Ca²⁺, and 10 Tris-Cl^–, pH 7.6. To prevent any endogenous production of ATP and to block all Na⁺ channels, 1 mM NaCl and 100 nM tetrodotoxin were always added to the external solutions. The osmolarity of all solutions was adjusted to 940 mosmol/l. At the beginning of the experiments, the axons were routinely dialyzed for 45 min with a standard medium containing 1 mM EGTA and free of Ca²⁺ and ATP. In all cases, each axon served as its own control because steady-state fluxes were measured before and after a given experimental condition. Reagents were obtained from Sigma Chemical (St. Louis, MO). Working temperature was between 17° and 18°C. SEA, provided by Taisho Pharmaceutical (Tokyo, Japan), was dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution and later diluted directly in the external or internal medium. The DMSO was always at or below 0.1%, a concentration without effect on the Na⁺/Ca²⁺ exchange fluxes.

	RESULTS

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Effect of extra- and intracellular SEA on different exchange modes of the Na⁺/Ca²⁺ exchanger in dialyzed axons containing physiological [Na⁺]_i and no ATP. Iwamoto et al. (14) reported that KB-R7943, an inhibitor of the Na⁺/Ca²⁺ exchanger weaker than SEA (17), preferentially inhibited the reverse exchange mode (12). In addition, these authors showed that KB-R7943 acts mostly from the extracellular side. On the other hand, SEA seems to act on the intracellular regulatory loop of the exchanger (17). To elucidate on which membrane side SEA works and to advance understanding of its mechanisms of action, we studied its effects in both extracellular and intracellular media on three modalities of the exchanger: forward (Na_o⁺/Ca_i²⁺), reverse (Ca_o²⁺/Na_i⁺), and Ca_o²⁺/Ca_i²⁺ exchanger modes. The basic dialysis solution contained a physiological [Na⁺]_i of 40 mM and a [Ca²⁺]_i of 5 µM, which is the K_m for the Ca_i²⁺ regulatory site at that [Na⁺]_i in the absence of ATP (6).

In the axon shown in Fig. 1 and in the presence of full [Na⁺]_o (440 mM) with no added Ca_o²⁺, the forward Na_o⁺/Ca_i²⁺ exchange reaches a value of 240 fmol·cm^–2·s^–1. Replacement of Na_o⁺ with Li⁺ in the presence of 3 mM Ca_o²⁺ induces a Ca²⁺/Ca_i²⁺ exchange of 125 fmol·cm^–2·s^–1. Upon addition of 3 µM external SEA, the Ca²⁺/Ca²⁺ exchange slowly drops by 50%. Upon returning to the initial conditions of 440 mM Na_o⁺ and 0 Ca_o²⁺, the forward Na⁺/Ca²⁺ exchange reached only 170 fmol·cm^–2·s^–1, indicating a 37% inhibition by SEA on this exchange mode. Figure 2 displays a similar protocol used to explore the reverse mode of the exchanger (Ca_o²⁺-dependent ²²Na efflux). In the presence of 440 mM Na⁺ and 0 Ca_o²⁺, the homologous Na_o⁺/Na_i⁺ exchange reached 12,000 fmol·cm^–2·s^–1; removal of Na_o⁺ and addition of 3 mM Ca_o²⁺ (a saturating concentration in the presence of full Li⁺) produced a reverse exchange of 5,500 fmol·cm^–2·s^–1. In this case, 3 µM extracellular SEA reduced the reverse exchange to 3,200 fmol·cm^–2·s^–1, corresponding to a 42% inhibition. In contrast, under conditions otherwise similar to those used before {40 mM [Na⁺]_i, 5 µM [Ca²⁺]_i, no ATP and intracellular pH (pH_i) 7.3}, Fig. 3 shows that 3 µM intracellular SEA reduced Na_o⁺-dependent ⁴⁵Ca²⁺ efflux (forward) from 230 to 50 fmol·cm^–2·s^–1, which amounts to 80% inhibition. This value is significantly higher than that observed when SEA was applied externally. A dose-response curve for cytosolic SEA inhibition of the forward Na⁺/Ca²⁺ exchange is shown in Fig. 4. The experimental points were fitted to a Michaelis-Menten equation, resulting in K_i of 1 ± 0.15 µM (n = 14). At the high ionic strength used in our dialysis solutions, this value is >50 times higher than that reported for mammalian tissues (17, 19, 25) (see DISCUSSION).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Effect of extracellular SEA0400 {SEA; 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline} on Nao+/Cai2+ (forward) and Cao2+/Cai2+ exchange. Closed circles correspond to the efflux of Ca2+ in the 440 mM Nao+/0 Cao2+ (forward exchange) or in 3 mM Ca2+-0 Nao+ (Ca2+/Ca2+ exchange). Open circles represent the efflux of Ca2 in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and units of concentration are indicated at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. SEA was added directly to the external medium from a 10 mM stock solution in dimethyl sulfoxide (DMSO). See MATERIALS AND METHODS.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effect of extracellular SEA on Nai+/Cao2+ exchange (reverse exchange). Closed circles correspond to the efflux of Na+ in 440 mM Nao+-0 Cao2+ (Na+/Na+ exchange) or in 3 mM Ca2+-0 Nao+ (reverse exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentration levels are shown at left, and the actual concentrations are indicated over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. SEA was added directly to the external medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (18K): [in this window] [in a new window]   Fig. 3. The effect of intracellular SEA on Nao+/Cai2+ (forward) exchange. Closed circles correspond to the efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are indicated at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Intracellular concentration dependence of SEA inhibition of Nao+/Cai2+ (forward) exchange. Data are from 14 squid axons showing the concentration dependence of intracellular SEA on Nao+-dependent Ca2+ efflux (forward exchange). The apparent inhibition constant for SEA was 1 ± 0.5 µM. Bars on the points represent SE. Numbers in parentheses refer to the number of axons.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Effect of intracellular SEA on Nao+/Cai2+ (forward) exchange in the absence of Nai+. Closed circles correspond to the efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are shown at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. Note the lack of SEA inhibition in the forward Na+/Ca2+ exchange in the absence of Nai+. Also note that the addition of 40 mM Nai+ in the presence of SEA rapidly led to inhibition of the flux, while removal of Nai+ reversed inhibition. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Ineffectiveness of intracellular SEA on the Nao+-Cai2+ (forward) exchange in the presence of ATP. Closed circles correspond to efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are shown at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. Note the negligible inhibition of forward Na+/Ca2+ exchange by SEA in the presence of internal ATP. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (21K): [in this window] [in a new window]   Fig. 11. A comprehensive kinetic model of ionic (Nai+, Hi+, and Cai2+) and metabolic (ATP and PA) modulation of the Na+/Ca2+ exchanger, together with the proposed sites of action of SEA. This exchange model has been proposed elsewhere (6, 7). The positive and negative signs represent the stabilization or destabilization of the carrier conformations induced by SEA and ATP, respectively (see DISCUSSION).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Ineffectiveness of intracellular SEA on the Nao+-Cai2+ (forward) exchange at alkaline pHi 8.5 in the presence of Nai+. Closed circles correspond to the efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are shown at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. Note the almost nonexistent inhibitory effect of SEA on the forward Na+/Ca2+ exchange at intracellular pH 8.5, even in the presence of Nai+ and with no ATP. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (16K): [in this window] [in a new window]   Fig. 10. pHi dependence of SEA inhibition of Nao+/Cai+ (forward) exchange in the presence and absence of Nai+. Data are from 15 squid axons showing the pHi dependence of 3 µM intracellular SEA inhibition of the Nao+-dependent Ca2+ efflux (forward exchange) in the presence () and absence () of Nai+. Bars on the points represent SE. Numbers in parentheses refer to the number of axons.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effect of intracellular SEA on the Nao+/Cai2+ (forward) exchange at acidic pHi 6.7 in the absence of Nai+. Closed circles correspond to the efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward) exchange. Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are indicated at left, and the actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. Note the large reduction in the forward Na+/Ca2+ exchange at pHi 6.7 and the significant inhibition of 3 µM SEA at this acidic pHi even with no Nai+. Also note that raising pHi to 7.3 completely reversed inhibition. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

View larger version (15K): [in this window] [in a new window]   Fig. 9. SEA inhibition is irreversible at acidic pHi even in the absence of Nai+. Closed circles correspond to the efflux of Ca2+ in 440 mM Nao+-0 Cao2+ (forward exchange). Open circles represent the efflux of Ca2+ in the absence of both Nao+ and Cao2+ (unspecific and/or leak fluxes). The inclusion and/or withdrawal of intra- and extracellular ligands are indicated at top. The ligands and concentrations are indicated at left, and actual concentrations are shown over the corresponding lines. Note that Li+ was used as an inert Na+ replacement. Note the low level of the forward Na+/Ca2+ exchange at intracellular pHi 6.7 and the significant inhibition of 3 µM SEA at this acidic pHi even with no Nai+. Also note that withdrawal SEA at a constant pH of 6.7 did not release inhibition. SEA was added directly to the internal dialysis medium from a 10 mM stock solution in DMSO. See MATERIALS AND METHODS.

	DISCUSSION

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In the present work, we have studied the effects of extra- and intracellular SEA on steady-state outward (forward exchange), inward (reverse exchange), and Ca²⁺/Ca²⁺ modes of the Na⁺/Ca²⁺ exchanger. Although previous studies with SEA revealed mostly changes in the peak outward Na⁺/Ca²⁺ exchange currents, the data also show that SEA inhibits steady-state currents (3). In our experiments, the enhancement by SEA of Na_i⁺ inhibition of the Na⁺/Ca²⁺ exchanger almost certainly reflects Na_i⁺ interaction at the Na⁺ inactivation site, because 1) Na_i⁺ inactivation is strongly increased by protons, 2) ATP and intracellular alkalinization relieve inhibition, 3) Na_i⁺ and H_i⁺ synergically promote SEA effects, and 4) internal acidification favors SEA inhibition even in the absence of Na_i⁺. In other words, the relationships between SEA inhibition and ligands interacting with the intracellular loop can be fully accounted for by the model recently proposed for regulation of the squid Na⁺/Ca²⁺ exchanger (6). The normal ATP concentration in a squid axon is 3–4 mM. Therefore, the inhibitory potency of SEA is expected to be largely diminished under physiological conditions. Actually, this hypothesis is in line with a recent work, published while this article was in preparation, in which inhibition by SN-6, a new benzyloxyphenyl derivative, in NCX1-transfected cells occurred in the inhibitory peptide region (related to Na_i⁺ inhibition) and was enhanced by intracellular ATP depletion (15). These results strongly support our proposed model in which the site of SEA inhibition is intracellular and acts through Na_i⁺-H_i⁺ synergy, which in turn is modulated by ATP level (Fig. 11),

Another interesting feature of our present results is that some characteristics of SEA inhibition described for NCX1 are not reproduced in squid axons. The inhibition does not occur mainly in the reverse exchange mode, but the three investigated modes (forward, reverse, and Ca²⁺/Ca²⁺ exchange) are affected. Also, we found no evidence that inhibition takes place preferentially from outside the membrane. To the contrary, the forward exchange is inhibited >80% from inside the axon, compared with only 40–50% from the external surface. As pointed out above, the data support the notion that SEA acts at or very near the intracellular loop of the exchanger (see Ref.3). The fact that the observed onset times of SEA inhibition of the forward Na⁺/Ca²⁺ exchange are not very different from those observed outside (Fig. 1) or inside the axon (Fig. 3) does not mean that the actual rates are the same. Figures 2 and 3 show that the washout of Na⁺ from the extracellular side takes place in <6 min. On the other hand, in the intracellular phase (see Fig. 5), that washout takes >20 min. These differences are due to geometry that makes the accessibility to extracellular Na⁺ sites easier than that associated with sites located inside the membrane. This point is also illustrated in Fig. 5, in which full stimulation of the exchanger by 3 mM ATP (>10 times the K_m) occurs after 30 min of its inclusion in the dialysis solution. Therefore, one should expect much faster inhibition of the exchanger by external application of SEA if the inhibitor is acting on an extracellular site. Nevertheless, we cannot rule out completely an additional external site involved in SEA inhibition. It could be argued that changes in pH_i may affect SEA inhibition through changes in its state of protonation. SEA is a 2,5-dimethoxyaniline. In general, the acidic dissociation constant (pK_a) of anilines is 4.0; actually, a compound with a closely related structure, the 3,5-dimethoxy aniline, has a pK_a of 3.82 (16). Consequently, at pH 6.7, practically all SEA are in a nonprotonated state. This makes it unlikely that a variation in pH_i explored in the present study, particularly alkalinization from pH 7.3 to 8.5 that prevents inhibition, will significantly modify the amount of unprotonated SEA molecules.

Under favorable conditions (with 40 mM Na_i⁺, pH 7.3, and no ATP or with pH_i 6.7, no ATP, and even without Na_i⁺), inhibition by SEA cannot be reversed for at least 1 h after its removal from the dialysis solution (see Fig. 9). One explanation for the irreversibility of SEA inhibition may be an actually very slow off rate constant for binding to the exchanger. Another alternative is that because of its known high hydrophobicity, it is very difficult to remove it from the lipid environment of the membrane; this requires a hydrophobic region around its actual binding site in the exchanger molecule. In addition, a nonspecific accumulation of SEA in glass pipettes and infusion tubes cannot be ruled out. On the other hand, removal of the cointeracting agent of inhibition, Na_i⁺ (Fig. 5) or H_i⁺ (alkalinization; see Fig. 8), even in the continuous presence of SEA, leads to a clear release of inhibition. The fact that the K_i for SEA at a high ionic strength of squid axons is 1 µM (>50 times higher than in mammalian NCX) makes it difficult to conclude that the absence of reversibility is due to the presence of an SEA concentration of 3 µM in the SEA-free solutions. The almost inescapable conclusion is that SEA binds quite tightly to the exchanger, and as a consequence of that binding, it facilitates the reversible attachment of the natural H_i⁺ + Na_i⁺ synergic inhibitors.

In summary, our data provide a new, extended explanation for the mechanisms underlying SEA inhibition of the Na⁺/Ca²⁺ exchanger. This compound not only enhances Na_i⁺-dependent inactivation but also stabilizes the conformation of the exchanger bound to the first proton. Further experiments are required to explore whether the ligand interactions regulated by phosphoarginine, too, are a target for SEA inhibition. We think that further investigations into the mechanism by which this novel inhibitor acts could produce beneficial pharmacological information and contribute to the understanding of the molecular basis for the regulation of the Na⁺/Ca²⁺ exchanger.

	GRANTS

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This work was supported by National Science Foundation Grant IBN-9631107, Agencia Nacional de Promoción Científica y Tecnológica-FONCYT-Argentina Grant PICT99 05-05158, and Consejo Nacional de Investigaciones Científicas y Tecnológicas Grant MCT-CONICIT-Venezuela S1-99000946 (Proyecto de Grupo IVIC-UCV-2001) and by Fundación Polar (Venezuela) and Fundaciencias (Mochima Edo Sucre).

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. DiPolo, Laboratorio de Permeabilidad Ionica, Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Apartado 21827, Caracas 1020A, Venezuela (E-mail: dipolor{at}ivic.ve)

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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