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J. Biol. Chem., Vol. 282, Issue 37, 26884-26896, September 14, 2007

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Indirect Activation of the Epithelial Na⁺ Channel by Trypsin^*

Abderrahmane Bengrine, Jinqing Li, L. Lee Hamm, and Mouhamed S. Awayda¹

From the Department of Physiology and Biophysics, State University of New York, Buffalo, New York 14214 and the Department of Medicine, Tulane University Health Sciences Center, New Orleans, Louisiana 70112

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 and subunits (ENaC). 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 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 ENaC, 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.

Received for publication, December 26, 2006 , and in revised form, June 25, 2007.

^* This work was supported by National Institutes of Health Grant DK55626 (to M. S. A.) and a Veterans Affairs Merit Award (to L. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, State University of New York, 124 Sherman Hall, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-3547; Fax: 716-829-2344; E-mail: awayda{at}buffalo.edu.

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