Abstract
We tested the hypothesis that the serine protease trypsin can indirectly activate the epithelial Na(+) channel (ENaC). Experiments were carried out in Xenopus oocytes and examined the effects on the channel formed by all three human ENaC subunits and that formed by Xenopus epsilon and human beta and gamma subunits (epsilonbetagammaENaC). Low levels of trypsin (1-10 ng/ml) were without effects on the oocyte endogenous conductances and were specifically used to test the effects on ENaC. Addition of 1 ng/ml trypsin for 60 min stimulated the amiloride-sensitive human ENaC conductance (g(Na)) by approximately 6-fold. This effect on the g(Na) was [Na(+)]-independent, thereby ruling out an interaction with channel feedback inhibition by Na(+). The indirect nature of this activation was confirmed in cell-attached patch clamp experiments with trypsin added to the outside of the pipette. Trypsin was comparatively ineffective at activating epsilonbetagammaENaC, a channel that exhibited a high spontaneous open probability. These observations, in combination with surface binding experiments, indicated that trypsin indirectly activated membrane-resident channels. Activation by trypsin was also dependent on catalytic activity of this protease but was not accompanied by channel subunit proteolysis. Channel activation was dependent on downstream activation of G-proteins and was blocked by G-protein inhibition by injection of guanyl-5'-yl thiophosphate and by pre-stimulation of phospholipase C. These data indicate a receptor-mediated activation of ENaC by trypsin. This trypsin-activated receptor is distinct from that of protease-activated receptor-2, because the response to trypsin was unaffected by protease-activated receptor-2 overexpression or knockdown.
Highlights
Activation of epithelial Na؉ channel (ENaC) by exogenous protease or by co-expression with proteases persists in heterologous expression systems such as Xenopus oocytes; differences are observed between systems in that many epithelial cells require prolonged prior incubation with a protease inhibitor before observing the effects of exogenous proteases
Both effects could be abolished by mutation of furin processing consensus sites on these ENaC subunits, indicating that channel cleavage through the biosynthetic pathway leads to its activation
These mutations did not affect the response of ENaC to trypsin, and this leaves the possibility that extracellular exogenous proteases may affect ENaC via different mechanisms than the cellular
Summary
Xenopus laevis Oocytes—Oocytes were prepared as previously described (19). Briefly, surgically removed oocytes were defoliculated in Ca2ϩ-free buffer containing 1 mg/ml collagenase (type 1A, Sigma). Trypsin was added to the external bath after the formation of a seal and establishing baseline channel activity This utilized a large tip electrode connected to an additional manipulator and delivered ϳ75 l of ND-94 solution containing 10 ng/ml trypsin onto the surface of the oocyte. The current signal was filtered at 200 Hz and digitally acquired at 1 KHz. ENaC currents were identified from their signature long open and closed times, single channel conductance, current-voltage relationship, and in some experiments partial block by low doses of amiloride. ENaC currents were identified from their signature long open and closed times, single channel conductance, current-voltage relationship, and in some experiments partial block by low doses of amiloride These currents were only observed in ENaC-expressing oocytes. Statistical significance was determined using Student’s t test at the 99% or 95% confidence levels
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