Defining an inhibitory domain in the α-subunit of the epithelial sodium channel

Abstract

Epithelial sodium channels (ENaC) are processed by proteases as they transit the biosynthetic pathway. We recently observed that furin-dependent processing of the α-subunit of ENaC at two sites within its extracellular domain is required for channel activation due to release of a 26-residue inhibitory domain. While channels with α-subunits lacking the furin sites are not cleaved and have very low activity, channels lacking the furin consensus sites as well as the tract between these sites (αD206–R231) are active. We analyzed channels with a series of deletions in the tract αD206–R231 and lacking the α-subunit furin consensus sites in Xenopus laevis oocytes. We found an eight-residue tract that, when deleted, restored channel activity to the level found in oocytes expressing wild-type ENaC. A synthetic peptide, LPHPLQRL, representing the tract αL211–L218, inhibited wild-type ENaC expressed in oocytes with an IC50 of 0.9 μM, and inhibited channels expressed in collecting duct cells and human primary airway epithelial cells with an IC50s of between ∼50 and 100 μM. Analyses of peptides with deletions within this inhibitory tract indicate that eight residues is the minimal backbone length that is required for ENaC inhibition. Analyses of 8-mer peptides with conserved and nonconserved substitutions suggest that L1, P2, H3, P4, and L8 are required for inhibitory activity. Our findings suggest that this eight-residue tract is a key conserved inhibitory domain that provides epithelial cells with a reserve of inactive channels that can be activated as required by proteases.

SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

“…It has been proposed that the extracellular loops of ENaC have a role in channel gating, and the ␣ and ␥ subunits of ENaC have been reported to contain short inhibitory segments that are removed during proteolytic cleavage to activate the channel (30,31). We speculate that ENaC subunits that have already been cleaved by extracellular serine proteases are likely to be SPLUNC1-insensitive.…”

The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

“…The inhibitory peptide derived from aENaC cleavage impairs NHK galvanotaxis Activation of the ENaC channel by proteases releases a short self-inhibitory sequence from the mouse aENaC of amino acids 182-190, LPHPLQRL (Carattino et al, 2006;Carattino et al, 2008). The cleaved ENaC channel is constitutively active and stays in the open state; treating mouse cortical collecting duct cells and human airway epithelial cells with synthetic LPHPLQRL peptide blocks the opening of ENaC (Carattino et al, 2006;Carattino et al, 2008).…”

“…The cleaved ENaC channel is constitutively active and stays in the open state; treating mouse cortical collecting duct cells and human airway epithelial cells with synthetic LPHPLQRL peptide blocks the opening of ENaC (Carattino et al, 2006;Carattino et al, 2008). We used this inhibitory peptide to further confirm the role of open ENaC channels in galvanotaxis.…”

“…To study if ENaC translocates to the cathodal edge of the cell during galvanotaxis, aENaC was immunolocalized in both NHK and MEK at different time points after exposure to the EF, using antibodies that detect all forms of aENaC: the fulllength 80-85 kDa and the cleaved, active 60-65 kDa (Fig. 4B) (Carattino et al, 2006;Carattino et al, 2008). At time zero, aENaC was randomly located at the cell periphery and in perinuclear areas (Fig.…”

“…Scrambled control peptide (QLHLLPPR) and ENaC inhibitory peptide (LPHPLQRL) (Carattino et al, 2006;Carattino et al, 2008) with acetylated and amidated ends were ordered (HPLC purified, purity .90%) from GenScript (Piscataway, NJ). NHKs were pre-treated with 20 mM peptides for 1 hour before the galvanotaxis assay.…”