Capsazepine Is a Novel Activator of the δ Subunit of the Human Epithelial Na+ Channel*

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as α, β, γ, and δ. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCδ, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCδβγ (hENaCδβγ) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of –60 mV in hENaCδβγ-expressing oocytes were markedly enhanced by the application of capsazepine (≥1 μm), and the capsazepine-induced current was mostly abolished by the addition of 100 μm amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 μm. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCδ homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCα and caused a slight decrease on the hENaCαβγ current. In conclusion, capsazepine acts on ENaCδ and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCδ.

The degenerin/epithelial Na ϩ channel superfamily has striking functional diversity including Na ϩ homeostasis, acid sensing, peptide-gating, acidosis-evoked nociception, and mechanotransduction (1)(2)(3)(4)(5). The amiloride-sensitive epithelial Na ϩ channel (ENaC) 1 is an essential control element for Na ϩ transport into cells and across epithelia. Four homologous ENaC subunits (␣, ␤, ␥, and ␦) have been cloned in mammals (6 -10). There is an overall ϳ37% amino acid identity between the ␣, ␤, ␥, and ␦ subunits. ENaC␣ is expressed mainly in epithelia, such as the kidney, lung, and colon, and binds with these ␤ and ␥ subunits to play a pathophysiological role (2,4), whereas the ␦ subunit is widely distributed throughout the brain and is also expressed in the heart, kidney, and pancreas (10,11). More recently, we have demonstrated that protons activate ENaC␦, indicating that it may contribute to pH sensation and/or pH regulation in the human brain (11). Most interestingly, the expressed sequence tag and genome project data bases show that an ENaC␦ gene has been found only in humans and chimpanzees (GenBank TM accession numbers U38254 and O46547, respectively), and there is no evidence of the orthologues in rats and mice. The corresponding genomic assignments of ENaC␦ were identified on human chromosome 1p36.3-p36.2 (12).
The functional expression and activity of ENaC are regulated by various endogenous and exogenous compounds. It has been described that aldosterone increases the transcription of serine/threonine kinase, and serum-and glucocorticoid-regulated kinase to reduce Nedd4-2-mediated degradation of ENaC␣␤␥, leading to the increased expression of ENaC␣␤␥ at the cell surface (13,14). Arachidonic acid reduces the surface expression of ENaC␣␤␥ while altering the rates of delivery and internalization of functional channels (15). In addition, syntaxin 1A interacts with cell surface ENaC␣␤␥ to rapidly decrease the open probability (16). On the other hand, ENaC␦␤␥ can bind with a copper transporter, Murr1, by interacting at the C-terminal domain of ENaC␦ to decrease the activity of the channel (17). In pharmacological profiles of ENaC, it is well known that the potassium-sparing diuretics, amiloride and benzamil, inhibit the activities of ENaC␣, ENaC␦, and the complexes with ␤ and ␥ subunits (6 -11). Extracellular protons reduce the activity of ENaC␣ (18), whereas ENaC␦ is activated by protons (11,19). In addition, ENaC␣␤␥ is activated by cAMP and blocked by Ni 2ϩ (20). In contrast to the relatively well documented ENaC␣␤␥ in pharmacological studies, it has been poorly investigated for ENaC␦. In particular, chemical activators for ENaC␦ have not yet been reported.
In this investigation, the effects of capsazepine on human ENaC (hENaC) current were examined by using electrophysiological analyses in the Xenopus oocytes expression system. Capsazepine, which possesses a vanilloid moiety, has been developed as a synthetic competitive antagonist for the transient receptor potential vanilloid subfamily 1 (TRPV1) (21)(22)(23)(24). Here we show that capsazepine activates hENaC␦␤␥ in a concentrationdependent manner, although other vanilloids (capsaicin, resiniferatoxin, and olvanil) were not affected on the hENaC␦␤␥ current. Most interestingly, capsazepine causes potentiation of the hENaC␦ current but not hENaC␣ activity. To our knowledge, capsazepine is the first agonist to activate ENaC␦.

EXPERIMENTAL PROCEDURES
Molecular Biology-All experiments were approved by the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences and were conducted in accordance with the Declaration of Helsinki. Human samples free from neurological disorders were taken within 24 h after death with permission from the families of the deceased. Ethical principles and considerations were observed regarding forensic and related research using human organs and fluids obtained from autopsies. Human tissues were rapidly frozen on dry ice immediately after their removal at autopsy and were kept at Ϫ80°C until use. The total RNA was extracted from homogenates of human tissues using ISOGEN (Nippon Gene, Tokyo, Japan), following digestion with RNasefree DNase (Promega, Madison, WI). The reverse transcription (RT) was performed as follows. We heated the reaction mixture of total RNA (3 g) and 500 ng of an oligo(dT) 12-18 primer (Invitrogen) in 8 l of diethyl bicarbonate-treated water at 70°C for 10 min, chilled it for 1 min, added 11 l of the reaction buffer (as a final concentration, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, and 0.5 mM individual dNTPs), and incubated it at 50°C for 2 min. The mixture was incubated at 50°C for 90 min after the addition of 200 units of SuperScript II reverse transcriptase (Invitrogen), and then heated at 70°C for 15 min. After this RT procedure, we added 80 l of distilled water to the reaction mixture and used it for PCR. To isolate the full-length ENaCs from human skin (for ␣, ␤, and ␥ subunits) or brain (for ␦ subunit) cDNA, oligonucleotide primers were designed as follows: for hENaC␣ (GenBank TM accession number X76180), ϩ, 5Ј-gaa ttc gcc gcc acc ATG GAG GGG AAC AAG CTG GAG GAG CAG GAC T-3Ј, and Ϫ, 5Ј-tct aga TCA GGG CCC CCC CAG AGG ACA GGT GGA GGA ACT GGC CCC T-3Ј; for hENaC␤ (X87159), ϩ, 5Ј-gat atc gcc gcc acc ATG CAC GTG AAG AAG TAC CTG CTG AAG GGC CT-3Ј, and Ϫ, 5Ј-tct aga TTA GAT GGC ATC ACC CTC ACT GTC AGA CTC GAT GAC GTC CA-3Ј; for hENaC␥ (U48937), ϩ, 5Ј-gaa ttc gcc gcc acc ATG GCC CCC GGG GAG AAG ATC AAA GCC A-3Ј, and Ϫ, 5Ј-tct aga TCA GAG CTC ATC CAG CAT CTG GGT ATC TGT GAG CT-3Ј; for hENaC␦ (U38254), ϩ, 5Ј-gaa ttc gcc gcc acc ATG GCT GAG CAC CGA AGC ATG GAC GGG AGA-3Ј, and Ϫ, 5Ј-tct aga TCA GGT GTC CAG AGT CTC AAG GGG CTG GGG CCC AGC CCA GCT-3Ј. The sequences indicated by lowercase letters are EcoRI (gaa ttc), EcoRV (gat atc), XbaI (tct aga) recognition sites, and the Kozak sequence (gcc gcc acc), which were added to the insert PCR products into vector DNA in the proper orientation and to promote effective translation, respectively. The RT reaction product (2 l) was amplified using GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) in a total volume of 25 l of a solution containing 15 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.2 mM individual dNTPs, 1.5 mM MgCl 2 , 15 pmol of sense and antisense primers for each, 10% dimethyl sulfoxide, and 2.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems). The thermal cycler program used for PCR amplification included a 0.5-min denaturation step at 94°C, a 0.5-min annealing step at 58°C, and a 2-min primer extension step at 72°C after a preheating step at 94°C for 9 min. The amplification was performed for 45 cycles. Thereafter, the reaction mixture was heated at 72°C for 15 min. The PCR products were separated on a 1% agarose gel in Tris acetate/EDTA buffer, recovered from the gel fragments, cloned, and sequenced.
Xenopus Oocyte Electrophysiology-Electrophysiological studies in Xenopus oocytes, using a two-electrode voltage clamp technique, were performed as described previously (11). In brief, cRNA(s) (2 ng for homomeric channel or each 0.02 ng for co-expression) was injected into Xenopus oocytes, whereas the control oocytes were injected with an equal volume of diethyl dicarbonate-treated water, described as native throughout. After injection, oocytes were incubated at 20°C in a recording solution supplemented with 20 units/ml penicillin G, 20 g/ml streptomycin, and either 10 (for hENaC␣ and ␣␤␥) or 100 M (for hENaC␦ and ␦␤␥) amiloride for 24 -48 h before electrophysiological recordings. The recording solution had an ionic composition of 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , and 5 mM HEPES. The pH of the solution was adjusted to 7.5 with NaOH. The recording solution for pH experiments was prepared with the equivalent MES (pK a ϭ 6.15) instead of HEPES (pK a ϭ 7.55) and adjusted to a suitable pH with either NaOH or HCl. All electrophysiological recordings were performed at a holding potential of Ϫ60 mV. The current-voltage relationships were measured by using a ramp protocol from Ϫ100 to 50 mV for FIG. 1. Activation of hENaC␦␤␥ current by capsazepine. Whole-cell currents were recorded at a holding potential of Ϫ60 mV in the Xenopus oocyte expression system using a two-electrode voltage clamp technique. A, hENaC␦␤␥-expressed oocyte possessed a larger inward current than a native oocyte. The larger current in an hENaC␦␤␥-expressing oocyte was mostly inhibited by 100 M amiloride (Ami). In an hENaC␦␤␥ oocyte, the application of 10 M capsazepine (CZ) enhanced the inward current, and this current increase was recovered by the removal of capsazepine. After washout for a few minutes, the re-addition of capsazepine caused similar current activation. B, the current-voltage relationships in the absence and presence of 10 M capsazepine in native and hENaC␦␤␥-injected oocytes are shown. The capsazepine stimulus enhanced the hENaC␦␤␥ activity at all voltages examined. Note that neither the application of capsazepine nor amiloride induced any current in a native oocyte. 30 s. The recording chamber was continuously perfused with solution at a flow rate of 5 ml/min. All electrophysiological experiments were carried out at room temperature (25 Ϯ 1°C).
Statistics-Pooled data are shown as the mean Ϯ S.E. Statistical significance between two groups and among groups was determined by Student's t test and Scheffé's test after one-way analysis of variance, respectively. Significant difference is expressed in the figures (** or ##, p Ͻ 0.01). The data of the relationship between either capsazepine or proton concentrations and current responses were fitted by using the following equation after normalization by the maximum current amplitude (see Figs. 3B and 6): where I max is the maximum amplitude of the capsazepine-or proton-evoked current; K d is the apparent dissociation constant of capsazepine or protons; [A] is the concentration of capsazepine or protons, and n is the Hill coefficient.

Activation of hENaC␦␤␥ Current by Capsazepine-
The effects of capsazepine, a well known competitive antagonist for TRPV1 (21)(22)(23)(24), on the hENaC␦␤␥ current were examined using a two-electrode voltage clamp technique in the Xenopus oocyte expression system. When hENaC␦␤␥ heteromultimer was expressed in Xenopus oocytes, an inward current was induced at a holding potential of Ϫ60 mV, and the current was mostly inhibited by 100 M amiloride (Fig. 1A). The mean amplitude of the amiloride-sensitive inward current in hENaC␦␤␥-expressing oocytes was 615 Ϯ 24 nA (n ϭ 54, p Ͻ 0.01 versus native of 1 Ϯ 1 nA, n ϭ 17). In hENaC␦␤␥-injected oocytes, the application of 10 M capsazepine was markedly increased in the inward current by 785 Ϯ 42 nA (n ϭ 26, p Ͻ 0.01). The current increase by capsazepine was recovered to the resting level by the removal of capsazepine in all hENaC␦␤␥expressing oocytes tested (n ϭ 26). After washout for a few minutes, the re-addition of capsazepine caused current activation with similar amplitude and kinetics to first challenge (n ϭ 6). The current-voltage relationship showed that the application of 10 M capsazepine enhanced the channel activity at all voltages examined in hENaC␦␤␥-expressed oocytes (n ϭ 4; Fig.  1B). On the other hands, in native oocytes, the application of 10 M capsazepine did not induce any current (n ϭ 14), because the capsazepine-induced currents were mediated by the activation of ENaC␦␤␥.
Effects of Amiloride on Capsazepine-induced Current-To confirm whether the capsazepine-induced current originated from ENaC␦␤␥ expression, the effects of amiloride, an inhibitor of ENaCs, on the inward current in the presence of capsazepine were examined. The 10 M capsazepine-induced current in hENaC␦␤␥-injected oocytes (1570 Ϯ 53 nA, n ϭ 26) was dramatically blocked by the addition of 100 M amiloride (88 Ϯ 1% decrease, n ϭ 26, p Ͻ 0.01 versus capsazepine) and, moreover, significantly inhibited the current to 194 Ϯ 20 nA (n ϭ 26, p Ͻ 0.01 versus the initial resting current of 785 Ϯ 33 nA; Fig. 2). In native oocytes, the current amplitudes after the application of Dose Dependence of Current Activation by Capsazepine-The concentration dependence of capsazepine-induced current was examined in hENaC␦␤␥-expressed oocytes. In hENaC␦␤␥-expressed oocytes, changing the concentration of capsazepine ranged from 0.01 to 100 M, which showed that the inward current was significantly increased by capsazepine at a concentration of 1 M and more (n ϭ 7, p Ͻ 0.05 versus control of 703 Ϯ 79 nA), and the enhancement was in a concentration-dependent manner (2346 Ϯ 153 nA at 100 M, n ϭ 7; Fig. 3). The EC 50 value of capsazepine on the inward currents was 7.8 M, and the Hill coefficient was 1.3.
Effects of Capsazepine-related Compounds on hENaC␦␤␥ Currents-Capsazepine possesses a vanilloid moiety, which is a key factor for the binding for TRPV1 (24 -26). To examine whether the opening action for ENaC␦␤␥ was shared by structurally related compounds of capsazepine, the effects of capsazepine-related compounds on the inward current were measured in hENaC␦␤␥-expressing oocytes. The change in current amplitude after exposure to the following compounds at a concentration of 10 -100 M for 5 min in hENaC␦␤␥-expressing oocytes is shown in Fig. 4: vanilloid compounds were 30 M capsaicin, 10 M resiniferatoxin, and 30 M olvanil; the antagonist of a nonselective cation channel containing TRPV1 was 30 M ruthenium red; and the molecule structurally similar to capsazepine was 100 M dopamine. Regardless of this, the inward current was markedly enhanced by 30 M capsazepine as shown in Fig. 3 (n ϭ 6, p Ͻ 0.01), and the effects of other compounds were not statistically significant (n ϭ 6 for each, p Ͼ 0.05).
Sensitization by Capsazepine on Proton-activated hENaC␦␤␥ Current-We reported previously (11) that ENaC␦ activity was enhanced by external protons in the Xenopus oocyte expression system. Therefore, the effects of capsazepine on proton-activated hENaC␦␤␥ current in Xenopus oocytes were examined. In hENaC␦␤␥-expressed oocytes, the gradual decrease in pH from 7.5 to 4.0 resulted in significantly increased currents in a proton concentration-dependent manner (2279 Ϯ 92 nA, pH 4.0, n ϭ 10, p Ͻ 0.01 versus control of 821 Ϯ 69 nA; Fig. 6A). The half-maximal pH for activation of the hENaC␦␤␥ current was 5.9 Ϯ 0.1, and the Hill coefficient was 0.35 Ϯ 0.02 (n ϭ 10). In the presence of 1 M capsazepine, which by itself had a very small effect on hENaC␦␤␥ current (by 144 Ϯ 20 nA, n ϭ 10; see Figs. 3 and 6B), the dose-response curve for protons on hENaC␦␤␥ currents was shifted to the left (half-maximal pH of 6.3 Ϯ 0.1 and Hill coefficient of 0.42 Ϯ 0.02, n ϭ 10). The maximum amplitude of the acid-evoked current in the presence of 1 M capsazepine (by 1502 Ϯ 102 nA, n ϭ 10) was not statistically significant with that in the absence of capsazepine (by 1458 Ϯ 117 nA, n ϭ 10, p Ͼ 0.05). Inversely, the dose response to capsazepine was strongly potentiated by weak acidification to pH 7.0 (Fig. 6B), which was a subthreshold concentration of protons on hENaC␦␤␥ current (by 25 Ϯ 4 nA, n ϭ 10; see Fig. 5A). The decrease in pH from 7.5 to 7.0 caused a left shift of the dose-response curve for capsazepine from the EC 50 value of 8.3 Ϯ 0.6 M (Hill coefficient of 1.4 Ϯ 0.1, n ϭ 10) to 2.4 Ϯ 0.2 M (Hill coefficient of 1.0 Ϯ 0.1, n ϭ 10). The maximum response of the capsazepine-induced current during the exposure to pH 7.0 (by 1574 Ϯ 122 nA, n ϭ 10) was not statistically significant to that in pH 7.5 medium (by 1566 Ϯ 130 nA, n ϭ 10, p Ͼ 0.05).

DISCUSSION
Amiloride-sensitive ENaCs, members of the degenerin/ ENaC superfamily, regulate essential control elements for Na ϩ homeostasis into cells and across epithelia. The heteromultimeric ENaC␣␤␥ complex is expressed mainly in epithelia such as the kidney, lung, and colon to play a pathophysiological role (2,4). More recently, we have shown that homomeric ENaC␦ is widely distributed throughout the brain and is activated by protons, indicating that it may act as a pH sensor in the human brain (11). In pharmacological aspects, the chemical agents for ENaC␣ are well documented, although those for ENaC␦ are largely unknown except for amiloride and benzamil as ENaC inhibitors. In particular, no chemical activators have been reported for ENaC␦ to date. In this investigation, the effects of capsazepine, a well known competitive TRPV1 antagonist (21-24), on hENaC␦␤␥ were examined by electrophysiological studies in the Xenopus oocytes expression system. We have found that the application of capsazepine activates hENaC␦␤␥ in a concentration-dependent manner, and the enhancement is sensitive to amiloride. The most interesting finding in this investigation is that capsazepine activated the hENaC␦ current but not hENaC␣ activity.
When the heteromultimeric hENaC␦␤␥ complex was expressed in Xenopus oocytes, the application of capsazepine at a concentration of 1 M and more was markedly increased by an inward current. Because the capsazepine-induced current was significantly abolished by the addition of 100 M amiloride, an inhibitor of ENaCs, in hENaC␦␤␥-expressing oocytes, and capsazepine-evoked currents were not observed in native oocytes, the capsazepine-induced currents originated from the ENaC␦␤␥ expression. The capsazepine-induced current was maintained at a steady level during capsazepine application in hENaC␦␤␥-expressed oocytes (see Fig. 2A). It is exciting that the chemical agonists for the ion channel induce sensitization or desensitization of its activity, such as capsaicin for TRPV1 (23) and 2-aminoethoxydiphenyl borate for TRPV1 and TRPV3 (27,28). After washout for a few minutes, the sequential challenge of capsazepine causes current activation to the same extent as the first trial in hENaC␦␤␥-expressing oocytes (see Fig. 1A). These results indicate that capsazepine did not induce sensitization and desensitization to the activity of hENaC␦␤␥ under these conditions. The sensitivity to capsazepine was increased in a weak acidic medium of pH 7.0. Protons are activated the hENaC␦ current (11,19), but pH 7.0 is the subthreshold concentration of protons in the hENaC␦␤␥ current. At this proton concentration, the dose response for capsazepine on the inward currents was enhanced, indicating that the effect of capsazepine was sensitized by the addition of protons. Inversely, the pH dependence FIG. 5. Selective activation of hENaC␦ not hENaC␣ by capsazepine. Effects of capsazepine on homomeric and heteromeric hENaC currents were examined in Xenopus oocytes expressed hENaC␣, ␣␤␥, ␦, or ␦␤␥. A, typical current traces of hENaC␣, ␣␤␥, ␦, and ␦␤␥ expressed in Xenopus oocytes are represented. Amiloride (100 M, Ami)-sensitive homomeric hENaC␦ current was activated by the application of 30 M capsazepine (CZ) as well as in hENaC␦␤␥-injected oocytes. In turn, the effects of capsazepine on another ENaC core unit, the ␣ subunit, which is sensitive to amiloride at the lower concentration of 10 M, were examined. Unexpectedly, in contrast to hENaC␦ and ␦␤␥, the application of 30 M capsazepine had no effect on any inward currents in an hENaC␣-expressing oocyte. A slight current decrease by 30 M capsazepine was observed in an hENaC␣␤␥-injected oocyte. B, the effects of capsazepine on these ENaC currents are summarized. The number of oocytes used is given in parentheses. The statistical significance of the difference is expressed as p Ͻ 0.01 (**) versus control. of hENaC␦␤␥ was shifted to the left by the lower concentration of capsazepine (1 M), which by itself had a very small effect on hENaC␦␤␥ current. Similar shifts in sensitivity by the mixture of two different ligands are observed in some ion channels such as TRPV1 (23,27). These results indicate that capsazepine acts synergistically with protons, an activating factor of ENaC␦. It is still unknown whether the mechanism of channel activation by capsazepine is similar to that by protons. The similarity to the responses to capsazepine and protons in ENaC␦ (activation by both stimuli) and ENaC␣ (partially inhibition) may be a key point for elucidating the mechanism underlying the channel modulation.
Capsazepine has been developed as a synthetic competitive antagonist at TRPV1 (21,22,24). In a variety of bioassays in vitro and in vivo, capsazepine is effective against both capsaicin-and resiniferatoxin-evoked responses (21)(22)(23)(24). Capsazepine at micromolar concentrations, which are necessary to inhibit capsaicin-evoked responses in most tissues, also blocks voltage-dependent calcium channels (29, 30) and voltage-dependent potassium channels (30) as well as nicotinic acetylcholine receptors (31). Furthermore, recent evidence suggests that capsazepine protects against neuronal injury caused by oxygen glucose deprivation by inhibiting hyperpolarization-activated nonspecific cation channel current (32) and inhibits the transient receptor potential melastatin subfamily 8 (33). In addition to its competitive antagonism of TRPV1 and its inhibitory actions on these channels, in this investigation we have clarified that capsazepine activates hENaC␦␤␥ with an EC 50 of 8 M, providing us with a novel target for drug development and screening in the degenerin/ENaC superfamily.
A vanilloid structure containing capsazepine is a key factor for acting on TRPV1 (24 -26). Therefore, it was examined whether the vanilloid structure contributed to the mechanism underlying the activation of hENaC␦␤␥. No activation was observed in response to vanilloid compounds (capsaicin, resiniferatoxin, and olvanil), an antagonist for nonselective cation channel (ruthenium red), and a molecule structurally similar to capsazepine (dopamine) at the concentration of 10 -100 M in hENaC␦␤␥-injected oocytes. In contrast, the application of capsazepine significantly activated the hENaC␦␤␥ current. These results suggest that ENaC␦␤␥ activation by capsazepine may be independent of the vanilloid structure.
The most interesting finding in this investigation was that capsazepine activated hENaC␦ but not hENaC␣. The homomeric hENaC␦, as well as hENaC␦␤␥, was activated by capsazepine, indicating that capsazepine acts directly on ENaC␦ itself. The extent to the activation in hENaC␦ alone (1.6 Ϯ 0.1-fold by 30 M capsazepine, n ϭ 5) was significantly smaller than that in hENaC␦␤␥ (2.6 Ϯ 0.2-fold, n ϭ 21, p Ͻ 0.05), suggesting that the accessory ␤ and/or ␥ subunit(s) may modulate the channel current activated by capsazepine. Another ENaC core unit, human ␣ subunit with 37% amino acid identity to ␦ subunit, failed to increase the current amplitude by the application of capsazepine, and unexpectedly, capsazepine caused a slight reduction of the hENaC␣␤␥ current. It has been described that there are differences in Na ϩ permeability (␣:␦ ϭ ϳ2:0.6 as I Li ϩ /I Na ϩ ) and the amiloride sensitivity (␣:␦ ϭ 0.1:2.6 M as IC 50 ) between ␣ and ␦ subunits (4, 6, 7, 10). Because the chemical agents influencing strongly either the ␣ or ␦ subunit have been poorly investigated, capsazepine is a potentially powerful tool for the electrophysiological analysis of ENaC␦ and the elucidation of the clinical ENaC␦ function in humans.
In conclusion, we found that capsazepine acts selectively on ENaC␦ rather than ENaC␣ and causes the activation of ENaC␦, showing that capsazepine is the first known activator for ENaC␦. In addition to the function of ENaC␦ as a pH sensor in human brain as described previously (11), this finding in our study provides a starting point for a number of exciting follow up investigations into the physiological and pathological roles of ENaC␦ in vitro and in vivo in humans.