The epithelial sodium channel (ENaC) is probably a heterotrimer with three well characterized subunits (αβγ). to the β- and γ-subunits of ENaC. The genes encoding δ-hENaC and α-hENaC are localized Idazoxan Hydrochloride on human chromosomes 1p36.3-p36.2 (25) and 12p13 (26 27 respectively. Thus α-hENaC and δ-hENaC are mapped to different chromosomes whereas β- and γ-hENaC are found within a common 400-kb fragment on chromosome 16p12 (28) and probably arise from gene duplication. Two splice variants of δ-ENaC have been described as follows: a shorter form with 638 amino acids (GI 34101282) originally cloned from a human kidney cDNA library (17) and a longer form with 704 amino acids (GI 21752051) originally cloned from human testis (29). In neuronal tissue the two isoforms have a cell-specific expression pattern (8). So far no functional differences have been observed between the two splice variants expressed in heterologous expression systems (10). In Idazoxan Hydrochloride this study we used the shorter δ-ENaC isoform which was the first one to be cloned (17). In heterologous expression systems δ-ENaC has functional similarities with α-ENaC. Isolated expression of δ-hENaC in oocytes results in small but significant amiloride-sensitive sodium currents (17). These currents are increased Idazoxan Hydrochloride by a factor of about 50 when δ-hENaC is usually co-expressed together with β-hENaC and γ-hENaC. In contrast co-expression of δβ- δγ- or αδ-subunits results in small amiloride-sensitive currents similar to those seen with the expression of δ-ENaC alone (17). These findings suggest that δ-ENaC preferentially assembles and functions as a δβγ-channel. The biophysical properties of the δβγ-hENaC channel are different from those of the αβγ-channel (17). δβγ-ENaC is usually more than an order of magnitude less sensitive to amiloride than αβγ-ENaC for which the IC50 for amiloride inhibition is about 100 nm (17 30 Additional pharmacological differences are the activating effect of capsazepine and icilin on δβγ-ENaC and its inhibition by Evans blue (21 33 34 Another difference is the higher single-channel Na+ conductance of δβγ-hENaC (～12 pS) compared with αβγ-hENaC (～5 pS) (17). Interestingly both channels have a similar single-channel conductance for Li+ (～7 pS). Thus δβγ-hENaC is more permeable for Na+ than for Li+ whereas αβγ-hENaC has a higher permeability for Li+ than for Na+. Finally δβγ-hENaC but not αβγ-hENaC has been reported to be activated Idazoxan Hydrochloride by extracellular protons and may contribute to pH sensing (18 Idazoxan Hydrochloride 35 36 There is recent evidence that proteases contribute to ENaC regulation by cleaving specific sites in the extracellular loops of the α- and γ-subunits but not the β-subunit (37-41). The channel is thought to be in its mature and active form in its cleaved state but there is evidence for the presence of both cleaved and noncleaved channels in the plasma membrane (42). Cleavage may activate the channel by changing its conformation probably by releasing inhibitory peptides from the extracellular loops of α- and γ-ENaC (43-45). Cleavage of the γ-subunit seems to be particularly important for channel activation by extracellular proteases (39 46 As far as we know it has not yet been shown whether the δ-subunit is also proteolytically processed and whether δβγ-ENaC can be proteolytically activated by exposing the channel to extracellular proteases. In this study we investigated the functional properties of αβγ- and δβγ-hENaC expressed in oocytes. Our starting point was the striking observation that this amiloride-sensitive whole-cell LIPG current (Δwere anesthetized in 0.2% MS222 (Sigma) and oocytes were Idazoxan Hydrochloride obtained by a partial ovariectomy. The oocytes were isolated from the ovarian lobes by enzymatic digestion at 19 °C on a rocking platform for 3-4 h with 600-700 units/ml type 2 collagenase from (CLS 2 Worthington) dissolved in calcium-free OR2 solution (in mm: NaCl 82.5 KCl 2 MgCl2 1 and HEPES 1 adjusted to pH 7.4 with Tris). Defolliculated stage V-VI oocytes were injected (Nanoject automatic injector Drummond Broomall PA) with an equal amount of cRNA per ENaC subunit (injected amounts of cRNA per ENaC subunit per oocyte (ng per subunit) are given under “Results” or in the physique legends). The cRNAs were dissolved in RNase-free water and the total volume injected into each oocyte was 46 nl. Injected oocytes were stored at 19 °C either in ND96 (high Na+) or in ND9 (low Na+). The latter solution contained (in mm) the following: NaCl 9 NMDG-Cl 87.