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

Abstract

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 ␣, ␤, ␥, and ␦. 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 ENaC␦, 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 ENaC␦␤␥ (hENaC␦␤␥) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of ؊60 mV in hENaC␦␤␥-expressing oocytes were markedly enhanced by the application of capsazepine (>1 M), and the capsazepine-induced current was mostly abolished by the addition of 100 M amiloride. The stimulatory effects of capsazepine on the inward current were concentrationdependent with an EC 50 value of 8 M. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaC␦ homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaC␣ and caused a slight decrease on the hENaC␣␤␥ current. In conclusion, capsazepine acts on ENaC␦ and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaC␦.

Small Molecule Activator of the Human Epithelial Sodium Channel

“…Openers of ␦ ENaC, another sodium-selective pore-forming channel, have recently been reported. Capsazepine, a nonspecific inhibitor of TRPV1, and icilin, an activator of TRPM8 temperature-sensitive nonselective cation channels, increased amiloride-sensitive current by 100 -200% (apparent EC 50 of 8 and 33 M, respectively) in Xenopus oocytes expressing ␦ or ␦␤␥ hENaC but not ␣ or ␣␤␥ hENaC (14,15). In addition, agents that activate ENaC by removing Na ϩ self-inhibition, the rapid, transient increase in inward current followed by a decline to a lower steady state level after removal of amiloride or switch from a low to high extracellular Na ϩ solution, have been reported, including Zn 2ϩ ions, the mercurial agent p-chloromercuribenzoate, and trypsin-like as well as furin serine proteases (16 -21).…”

Icilin Activates the δ-Subunit of the Human Epithelial Na+ Channel

“…More recently, we have shown that 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 (Yamamura et al, 2004b). In addition, we have demonstrated that capsazepine is the first chemical agonist for ENaC␦ (Yamamura et al, 2004a). In this investigation, we have found that the application of icilin, which is a tetrahydropyrimidine-2-one derivative unrelated structurally to capsaz- Fig.…”

“…At this proton concentration, the dose-response for icilin on the inward currents was enhanced, indicating that the effect of icilin was sensitized by the addition of protons. Moreover, the concentration-dependence of hENaC␦␤␥ for icilin was shifted to the left by the lower concentration of capsazepine (1 M), which by itself had a very small effect on hENaC␦␤␥ current (Yamamura et al, 2004a). Similar shifts in sensitivity by the mixture of two different ligands were observed: protons/capsaicin, capsaicin/2-aminoethoxydiphenyl borate, and protons/2-aminoethoxydiphenyl borate in transient receptor potential vanilloid subfamily 1 (Caterina et al, 1997;Hu et al, 2004) as well as protons/capsazepine in ENaC␦␤␥ (Yamamura et al, 2004a).…”

“…In pharmacological profiles of ENaC␦, it is well-published that the potassium-sparing diuretics, amiloride and benzamil, inhibit the activities of ENaC␦ homomer and the heteromeric complexes with ␤ and ␥ subunits (Waldmann et al, 1995;. More recently, we have shown that capsazepine, which was originally developed as a competitive antagonist for transient receptor potential vanilloid subfamily 1 (Bevan et al, 1992;Szallasi et al, 1993;Caterina et al, 1997;Szallasi and Blumberg, 1999), is the first known chemical activator of ENaC␦ (Yamamura et al, 2004a).…”

“…The full-length hENaC␣ (GenBank accession number X76180), hENaC␤ (X87159), hENaC␥ (U48937), and hENaC␦ (U38254) were isolated from human skin (for ␣, ␤, and ␥ subunits) or brain (for ␦ subunit) cDNA, as described previously (Yamamura et al, 2004a).…”