Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, C34

Oral Communications

Identification of two cleavage sites in the γ-subunit of the epithelial sodium channel (ENaC) with functional importance for its proteolytic activation by plasmin

S. Haerteis1, M. Krappitz1, A. Diakov1, R. Rauh1, C. Korbmacher1

1. Institut f


Proteolytic processing of ENaC is essential for channel activation [1]. Recently, we reported that plasmin can proteolytically activate ENaC which may contribute to renal sodium retention in nephrotic syndrome [2]. In mouse γENaC a putative plasmin cleavage site (K194) has been reported [3]. For rat ENaC an indirect stimulatory effect of plasmin via prostasin has been proposed [4]. The aim of this study was to identify cleavage sites in human γENaC that are functionally important for channel activation by plasmin. Sequence comparison of human and mouse ENaC suggested a putative plasmin cleavage site in human γENaC (K189). To study its functional relevance we generated a γK189A mutant by site-directed mutagenesis and expressed wild-type (wt) and αβγK189A-ENaC in Xenopus laevis oocytes. The γK189A mutation reduced but did not abolish the stimulatory effect of plasmin (10 µg/ml) on ENaC. In contrast, mutating a putative prostasin site (γRKRK178AAAA) had no apparent effect on the stimulatory response to plasmin. Interestingly, a double mutation (γRKRK178AAAA; K189A) abolished the stimulatory effect of plasmin and also significantly reduced the stimulatory effect of chymotrypsin (2 µg/ml). Using a biotinylation approach, we investigated the appearance of ENaC cleavage products at the cell surface of wt or double mutant ENaC expressing oocytes treated with chymotrypsin for 5, 30, or 60 min. The time-dependent appearance of cleavage products correlated well with the stimulatory effect of chymotrypsin on ENaC currents and was significantly delayed in oocytes expressing the double mutant ENaC. Delayed proteolytic activation of the mutant channel was confirmed in single-channel recordings from outside-out patches. These experiments also demonstrated that the double mutation did not affect the single-channel conductance of ENaC. Moreover, they demonstrated that the activation of both wt and mutant channel by chymotrypsin was caused by a stepwise recruitment of so-called near silent channels. In summary, the reduced stimulatory effect of plasmin on αβγK189A-ENaC indicates that γK189 is a relevant plasmin cleavage site in human γENaC. However, the lack of a complete inhibition is in contrast to findings in mouse ENaC and suggests that an additional cleavage site is involved in the activation of human ENaC by plasmin. This is supported by our finding that a combination of the K189A and the RKRK178AAAA mutation abolished the stimulatory effect of plasmin. We conclude that in addition to the putative plasmin site a putative prostasin cleavage site is involved in mediating the proteolytic activation of human ENaC by plasmin.

Where applicable, experiments conform with Society ethical requirements