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J. Biol. Chem., Vol. 281, Issue 14, 9110-9117, April 7, 2006

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Regulation of Epithelial Na⁺ Channels (ENaC) by Methylation

A NOVEL METHYLTRANSFERASE STIMULATES ENaC ACTIVITY^*

From the Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Aldosterone acts to increase apical membrane permeability by activation of epithelial Na⁺ channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na⁺ transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na⁺ transport induced by aldosterone at early time points (1–4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.

Received for publication, August 22, 2005 , and in revised form, January 9, 2006.

^* This work was supported by National Institutes of Health Grants RO1DK47874 and DK57718 and by research funds from Dialysis Clinics Inc. 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: University of Pittsburgh Medical Center, 935 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15213-2550. Tel.: 412-647-7157; Fax: 412-647-6222; E-mail: Johnson{at}dom.pitt.edu.

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				W. G. Hill, M. B. Butterworth, H. Wang, R. S. Edinger, J. Lebowitz, K. W. Peters, R. A. Frizzell, and J. P. Johnson The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells J. Biol. Chem., December 28, 2007; 282(52): 37402 - 37411. [Abstract] [Full Text] [PDF]