HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 19/1/142 04-2314fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kunzelmann, K. Articles by Schreiber, R. Search for Related Content PubMed PubMed Citation Articles by Kunzelmann, K. Articles by Schreiber, R.

Purinergic inhibition of the epithelial Na⁺ transport via hydrolysis of PIP₂

K. Kunzelmann^*,1, T. Bachhuber^*, R. Regeer, D. Markovich, J. Sun and R. Schreiber^*

^* Institut für Physiologie, Universität Regensburg, Regensburg, Germany; and
Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia

¹Correspondence: Institut für Physiologie, Universität Regensburg, Universitätsstrasse 31, Regensburg, Germany. E-mail: uqkkunze{at}mailbox.uq.edu.au

SPECIFIC AIMS

We examined inhibition of amiloride sensitive Na⁺ absorption (I_sc-Amil) in native trachea and mouse collecting duct (M1) cells by stimulation of purinergic receptors. We tried to uncover the molecular mechanism by which hydrolysis of phosphatidylinositol bisphosphate (PIP₂) during stimulation of P2Y₂ receptors inactivates the epithelial Na⁺ channel ENaC, which is in charge of Na⁺ absorption.

PRINCIPAL FINDINGS

1. Stimulation of apically located purinergic receptors by ATP inhibits amiloride sensitive Na⁺ absorption in airway epithelia and in mouse collecting duct cells
The aminoglycoside neomycin, which binds with high affinity to PIP₂, suppressed inhibition of Na⁺ absorption by ATP.

2. Inhibition of the phosphatidylinositol 4 (PI₄) kinase by wortmannin (10 µmol/L) or blocking the diacylglycerol kinase with R59022 abolished recovery of Na⁺ absorption from inhibition by ATP (Fig. 1 )
In contrast, inhibition of phospholipase C (PLC) with edelfosine (ET-18-OCH3) blocked the inhibitory effect of ATP on Na⁺ absorption.

View larger version (12K): [in this window] [in a new window]   Figure 1. Summary of experiments demonstrating inhibition of Isc-Amil by ATP and recovery after 90 min. Application of wortmannin (15 min, 10 nmol/L) reduces Isc-Amil and abolishes recovery from ATP-inhibition. *significant difference when compared with precontrol (paired t test). Numbers in parentheses indicate number of experiments.

3. Attenuated luminal fluorescence by the PIP₂-binding pleckstrin homology domain fused to GFP (PLC₃-GFP) by stimulation with ATP, suggested depletion of apical PIP₂ pools, in parallel to a lack of recovery of Na⁺ absorption from inhibition by ATP

4. The ß subunit of ENaC was coimmunoprecipitated with PIP₂
Stimulation of P2Y₂-receptors by ATP inhibited ENaC when coexpressed in Xenopus oocytes. Hydrolysis of PIP₂ by phosphatidylinositol specific phospholipase C from Bacillus cereus and by incubation with the membrane permeable direct activator of phospholipase C, m-3M3FBS, also inhibits ENaC. Incubation of ENaC-expressing oocytes with wortmannin, injection of neomycin, and incubation at pH 6.0 all reduced the amiloride sensitive conductance.

5. Mutations in the putative PIP₂ binding domain of the ß subunit of ENaC abolished amiloride sensitive Na⁺ conductance, while mutation in the putative PIP₂ binding domain of the subunit were without significant effects on the Na⁺ conductance (Fig. 2 )

View larger version (17K): [in this window] [in a new window]   Figure 2. Summary of amiloride sensitive whole cell conductances (GAmil) measured Xenopus oocytes expressing ,ß,-wtENaC or ENaC in which either the ß subunit (ß2N, ß4N) or the subunit (2N, 4N) have been mutated. wt,ßwt,2N-ENaC (2N, 2 lysines have been replaced by nonpolar amino acids) and wt,ßwt,4N-ENaC (4N, 4 lysines have been replaced by nonpolar amino acids) produce conductances indistinguishable to that of ,ß,-wtENaC. Conductances produced by the double mutant wt,ß2N,wt-ENaC (ß2N) were attenuated and were almost abolished for the quadruple mutant wt,ß4N,wt-ENaC (ß4N). *Significant difference from ßwt (unpaired t test). Numbers in parentheses indicate number of experiments.

6. The membrane expression of ENaC was not affected by mutations in ß-ENaC as demonstrated by FITC fluorescence and chemiluminescence of FLAG tagged ENaC subunits

CONCLUSIONS AND SIGNIFICANCE

The present data demonstrate that the physiologically relevant inhibition of epithelial Na⁺ absorption by stimulation of purinergic receptors is related to PIP₂ hydrolysis. An N-terminal domain located in the ß subunit of ENaC binds to phosphatidylinositol bisphosphate. This PIP₂ binding domain in ß-ENaC is formed by clustered, positively charged residues and probably mediates electrostatic interaction with the negatively charged phosphates of PIP₂. PIP₂ binding to ß-ENaC is necessary to maintain Na⁺ channel activity. Results from the present study suggest that ATP binding to P2Y₂ receptors leads to hydrolysis of PIP₂, which is paralleled by reduced channel activity rather than changes in surface expression. Thus the COOH terminus of ENaC determines surface expression of the channel, while the NH₂ terminus regulates channel activity, probably by controlling the single channel open probability. Since ENaC has been shown to be located in lipid rafts, further studies will have to demonstrate whether functional interaction between purinergic receptors and ENaC takes place in these membrane microdomains. The present findings may be of potential interest for a novel pharmacological approach to cystic fibrosis lung disease which is caused in part by excessive Na⁺ absorption in the airways. Compounds which interfere with the PIP₂ metabolism and deplete airway epithelial cells of PIP₂ may be useful for deactivation of ENaC and inhibition of Na⁺ absorption.

View larger version (36K): [in this window] [in a new window]   Figure 3. Model for the purinergic regulation of the epithelial Na+ channel ENaC. A) Under resting conditions P2Y2 receptors are not activated and the inner leaflet of the lipid bilayer contains a high concentration of PIP2. N-terminal binding of ß-ENaC to PIP2 maintains the Na+ channel activity. B) Stimulation of purinergic receptors by ATP results in activation of the phospholipase C and hydrolysis of PIP2. Lack of PIP2 binding to the N-terminus of ß-ENaC leads to a change in protein conformation and reduced channel activity by a reduction in the open probability.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2314fje;

This article has been cited by other articles:

				O. Pochynyuk, V. Bugaj, A. Vandewalle, and J. D. Stockand Purinergic control of apical plasma membrane PI(4,5)P2 levels sets ENaC activity in principal cells Am J Physiol Renal Physiol, January 1, 2008; 294(1): F38 - F46. [Abstract] [Full Text] [PDF]

				V. Vallon P2 receptors in the regulation of renal transport mechanisms Am J Physiol Renal Physiol, January 1, 2008; 294(1): F10 - F27. [Abstract] [Full Text] [PDF]

				K. M. Weixel, R. S. Edinger, L. Kester, C. J. Guerriero, H. Wang, L. Fang, T. R. Kleyman, P. A. Welling, O. A. Weisz, and J. P. Johnson Phosphatidylinositol 4-Phosphate 5-Kinase Reduces Cell Surface Expression of the Epithelial Sodium Channel (ENaC) in Cultured Collecting Duct Cells J. Biol. Chem., December 14, 2007; 282(50): 36534 - 36542. [Abstract] [Full Text] [PDF]

				T. Rieg, R. A. Bundey, Y. Chen, G. Deschenes, W. Junger, P. A. Insel, and V. Vallon Mice lacking P2Y2 receptors have salt-resistant hypertension and facilitated renal Na+ and water reabsorption FASEB J, November 1, 2007; 21(13): 3717 - 3726. [Abstract] [Full Text] [PDF]

				L. Li, C. S. Wingo, and S.-L. Xia Downregulation of SGK1 by nucleotides in renal tubular epithelial cells Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1751 - F1757. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, Q. Tong, J. Medina, A. Vandewalle, A. Staruschenko, V. Bugaj, and J. D. Stockand Molecular Determinants of PI(4,5)P2 and PI(3,4,5)P3 Regulation of the Epithelial Na+ Channel J. Gen. Physiol., September 24, 2007; 130(4): 399 - 413. [Abstract] [Full Text] [PDF]

				A. Bengrine, J. Li, L. L. Hamm, and M. S. Awayda Indirect Activation of the Epithelial Na+ Channel by Trypsin J. Biol. Chem., September 14, 2007; 282(37): 26884 - 26896. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, Q. Tong, A. Staruschenko, and J. D. Stockand Binding and direct activation of the epithelial Na+ channel (ENaC) by phosphatidylinositides J. Physiol., April 15, 2007; 580(2): 365 - 372. [Abstract] [Full Text] [PDF]

				B. Button, M. Picher, and R. C. Boucher Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia J. Physiol., April 15, 2007; 580(2): 577 - 592. [Abstract] [Full Text] [PDF]

				A. Bengrine, J. Li, and M. S. Awayda The A-kinase anchoring protein 15 regulates feedback inhibition of the epithelial Na+ channel FASEB J, April 1, 2007; 21(4): 1189 - 1201. [Abstract] [Full Text] [PDF]

				O. Pochynyuk, Q. Tong, A. Staruschenko, H.-P. Ma, and J. D. Stockand Regulation of the epithelial Na+ channel (ENaC) by phosphatidylinositides Am J Physiol Renal Physiol, May 1, 2006; 290(5): F949 - F957. [Abstract] [Full Text] [PDF]

				R. Tarran, L. Trout, S. H. Donaldson, and R. C. Boucher Soluble Mediators, Not Cilia, Determine Airway Surface Liquid Volume in Normal and Cystic Fibrosis Superficial Airway Epithelia J. Gen. Physiol., April 24, 2006; 127(5): 591 - 604. [Abstract] [Full Text] [PDF]

				M. A. Gray Primary cilia and regulation of renal Na+ transport. Focus on "Heightened epithelial Na+ channel-mediated Na+ absorption in a murine polycystic kidney disease model epithelium lacking apical monocilia" Am J Physiol Cell Physiol, April 1, 2006; 290(4): C947 - C949. [Full Text] [PDF]

				O. Pochynyuk, A. Staruschenko, Q. Tong, J. Medina, and J. D. Stockand Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel J. Biol. Chem., November 11, 2005; 280(45): 37565 - 37571. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 19/1/142 04-2314fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kunzelmann, K. Articles by Schreiber, R. Search for Related Content PubMed PubMed Citation Articles by Kunzelmann, K. Articles by Schreiber, R.