Regulation of the epithelial Na+ channel by peptidases

Abstract

Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na(+) channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na(+) transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na(+) transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na(+) transport when coexpressed with ENaC in oocytes. The amphibian enzyme's apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na(+) hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung.

Figure 1

Proteolytic regulation of ENaC. The ENaC ααβγ heterotetramer is thought to be assembled initially in the endoplasmic reticulum. The N-terminal and C-terminal ends of each subunit reside in the cytosol. (Step 1) Portions of the extracellular domains of α- and γ-subunits are nicked by the transmembrane peptidase furin, probably in the lumen of trans-Golgi network. These intracellular processing events may increase ion transport activity when ENaC is inserted into the plasma membrane (Step 2). As shown in Step 3, trypsin-family “CAPs” upregulate Na⁺ transport via ENaC. These interactions probably are extracellular, as depicted. Known ENaC-activating peptidases include the type I transmembrane peptidase prostasin, shown here attached to the plasma membrane via a GPI lipid anchor. Certain type II transmembrane peptidases, including TMPRSS3 shown here, also activate ENaC. In addition, some free trypsin-like serine peptidases, notably trypsin itself, can increase ENaC-mediated Na⁺ transport. The target of these peptidases is not known; it may be ENaC or a protein that regulates ENaC. The amount of functional ENaC on the cell surface can be decreased by endocytic uptake (Step 4). A portion of endocytosed ENaC may be subject to further processing and recycled to the membrane (Step 5). Otherwise, ENaC in endosomes is likely to be ubiquinated, thereby tagging it for denaturation and total destruction by the peptidases and other proteins associated with the cytosolic proteasome (Step 6).