Thumb domains of the three epithelial Na+ channel subunits have distinct functions.

The epithelial Na+ channel (ENaC) possesses a large extracellular domain formed by a ß-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, ß, or γ). Removing the γ subunit thumb domain had no effect on Na+ currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or ß subunit thumb domain exhibited significantly reduced Na+ currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na+ self-inhibition response, whereas this response was retained in channels lacking a ß thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature ß subunit and mature γ subunit was observed when the ß or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na+ Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity.

Na+ inhibits the epithelial Na+ channel by binding to a site in an extracellular acidic cleft.

The epithelial Na(+) channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na(+), Cl(-), protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na(+) concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na(+) binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na(+). Mutations at selected sites altered the cation inhibitory preference to favor Li(+) or K(+) rather than Na(+). Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na(+). Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family.

Brief treatment with heparin-binding EGF-like growth factor, but not with EGF, is sufficient to accelerate epithelial wound healing.

BACKGROUND: Heparin-binding EGF-like growth factor (HB-EGF) contains, in contrast to EGF, a domain that binds to negatively charged glycans on cell surfaces and in extracellular matrix. We speculated that a short exposure to HB-EGF induces prolonged biological effects such as healing of wounds after immobilization in tissues. METHODS: Epithelial cell sheets in tissue and corneas in organ culture were treated briefly with HB-EGF or EGF and binding of the growth factors, time course of activation of the EGF receptor, and healing of wounds were compared. RESULTS: Treating human corneal epithelial cells for 2 min with HB-EGF resulted in 8h of detectable activation of the EGF receptor, but activation was much shorter after EGF treatment. A brief treatment with HB-EGF, but not with EGF, induced significant acceleration of healing in wounds in epithelial sheets in tissue and organ culture. Bound HB-EGF was detectable up to 16 h after brief treatments. Neutralizing antibodies added after HB-EGF treatment blocked acceleration of healing, demonstrating the role of bound HB-EGF in accelerating healing. CONCLUSIONS: A brief exposure to HB-EGF, but not to EGF, is sufficient to induce prolonged activation of the EGF receptor and to enhance healing. GENERAL SIGNIFICANCE: Bound HB-EGF can serve as a pool that induces prolonged activation of the EGF receptor. EGF has been used experimentally to treat poorly healing wounds, but the frequent applications that are necessary have hampered its use clinically. The findings imply that HB-EGF may be a useful long-acting alternative to EGF.

Multiple residues in the distal C terminus of the α-subunit have roles in modulating human epithelial sodium channel activity.

Epithelial sodium channels (ENaC) are critically important in the regulation of ion and fluid balance in both renal and respiratory epithelia. ENaC functional polymorphisms may contribute to alterations in blood pressure in the general population. We previously reported that the A663T polymorphism in the C terminus of the α-subunit altered ENaC functional and surface expression in Xenopus laevis oocytes (Samaha FF, Rubenstein RC, Yan W, Ramkumar M, Levy DI, Ahn YJ, Sheng S, Kleyman TR. J Biol Chem 279: 23900-23907, 2004). We examined whether sites in the vicinity of 663 influenced channel activity by performing scanning Ala mutagenesis. Interestingly, only αT663/G667Aßγ channels exhibited increased currents compared with αT663ßγ. This increase in channel activity reflected an increase in channel open probability and not an increase in channel surface expression. In contrast, decreases in channel activity were observed with both αT663/C664Aßγ and αT663/C664Mßγ channels. The decrease in functional expression of αT663/C664Mßγ channels correlated with decreased surface expression, suggesting that the αC664M mutation altered the intracellular trafficking of the channel. While cytoplasmic Cys residues may be modified by the addition of palmitate, we did not observe palmitoylation of αC664. Our results suggest that multiple residues in the distal part of the cytoplasmic C terminus have roles in modulating channel activity.

Pyk2 activation triggers epidermal growth factor receptor signaling and cell motility after wounding sheets of epithelial cells.

Activation of the epidermal growth factor receptor (EGFR) is a key signaling event that promotes cells to move and cover wounds in many epithelia. We have previously shown that wounding activates the EGFR through activation of the Src family kinases (SFKs), which induce proteolytic shedding of epidermal growth factor-like ligands from the cell surface. A major goal in wound healing research is to identify early signals that promote motility, and here we examined the hypothesis that members of the focal adhesion kinase family are upstream activators of the SFKs after wounding. We found that focal adhesion kinase is not activated by wounding but that a different family member, Pyk2 (PTK2B/RAFTK/CAKbeta), is activated rapidly and potently. Pyk2 interaction with c-Src is increased after wounding, as determined by co-immunoprecipitation experiments. Disruption of Pyk2 signaling either by small interfering RNA or by expression of a dominant negative mutant led to inhibition of wound-induced activation of the SFKs and the EGFR, and conversely, overexpression of wild-type Pyk2 stimulated SFK and EGFR kinase activities in cells. In wound healing studies, Pyk2 small interfering RNA or dominant negative inhibited cell migration. These results show that activation of Pyk2 is an early signal that promotes wound healing by stimulating the SFK/EGFR signaling pathway.

Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor.

Wounding usually causes considerable cell damage, and released ATP promotes migration of nearby epithelium. ATP binds to purinergic receptors on the cell surface and induces transactivation of the EGF receptor through signaling by the Src family kinases (SFKs). Here we tested whether ATP activates these kinases through Pyk2, a member of the focal adhesion kinase family. Pyk2 was rapidly and potently activated by treating corneal epithelial cells with ATP, and physical interaction of Pyk2 with the SFKs was enhanced. Disruption of Pyk2 signaling either by siRNA or by expression of a dominant-negative mutant led to inhibition of ATP-induced activation of the SFKs and the EGF receptor. Inhibiting Pyk2 activity also blocked ATP stimulation of healing of wounds in epithelial cell sheets. These data suggest that ATP stimulates sequential activation of Pyk2, SFKs, and the EGF receptor to induce cell migration.

Free edges in epithelial cell sheets stimulate epidermal growth factor receptor signaling.

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell-cell interaction but rather due to loss of physical constraints.