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Vol. 25, No. 1, 2010
Issue release date: 2010
Cell Physiol Biochem 2010;25:145–158
(DOI:10.1159/000272059)

Functional Characterization of a Partial Loss-of-Function Mutation of the Epithelial Sodium Channel (ENaC) Associated with Atypical Cystic Fibrosis

Huber R.1 · Krueger B.1 · Diakov A.1 · Korbmacher J.1 · Haerteis S.1 · Einsiedel J.2 · Gmeiner P.2 · Azad A.K.3 · Cuppens H.3 · Cassiman J.-J.3 · Korbmacher C.1 · Rauh R.1
1Institut für Zelluläre und Molekulare Physiologie and2Department Chemie und Pharmazie, Emil Fischer Centrum, Universität Erlangen-Nürnberg,3Center of Human Genetics, KU Leuven
email Corresponding Author

Abstract

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (Po). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a Po of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average Po of ENaC by ñ75%. Na+ self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel Po. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients.


 Outline


 goto top of outline Key Words

  • Xenopus laevis oocytes
  • Electrophysiology
  • Heterologous expression
  • Ion channel

 goto top of outline Abstract

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (Po). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a Po of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average Po of ENaC by ñ75%. Na+ self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel Po. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients.

Copyright © 2010 S. Karger AG, Basel


 goto top of outline Author Contacts

Prof. Dr. med. Christoph Korbmacher
Institut für Zelluläre und Molekulare Physiologie
Waldstr. 6, D-91054 Erlangen (Germany)
Tel. +49 9131 8522301, Fax: +49 9131 8522770
E-Mail christoph.korbmacher@physiologie2.med.uni-erlangen.de


 goto top of outline Article Information

Accepted: September 08, 2009
Published online: December 22, 2009
Number of Print Pages : 14


 goto top of outline Publication Details

Cellular Physiology and Biochemistry (International Journal of Experimental Cellular Physiology, Biochemistry andPharmacology)

Vol. 25, No. 001, Year 2010 (Cover Date: 2010)

Journal Editor: F. Lang, Tübingen
ISSN: 1015–8987 (Print), eISSN: 1421–9778 (Online)

For additional information: http://www.karger.com/journals/cpb


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Loss-of-function mutations of the epithelial sodium channel (ENaC) may contribute to pulmonary symptoms resembling those of patients with atypical cystic fibrosis (CF). Recently, we identified a loss-of-function mutation in the α-subunit of ENaC (αF61L) in an atypical CF patient without mutations in CFTR. To investigate the functional effect of this mutation, we expressed human wild-type αβγ-ENaC or mutant α F61Lβγ-ENaC in Xenopus laevis oocytes. The αF61L mutation reduced the ENaC mediated whole-cell currents by ñ90%. In contrast, the mutation decreased channel surface expression only by ñ40% and did not alter the single-channel conductance. These findings indicate that the major effect of the mutation is a reduction of the average channel open probability (Po). This was confirmed by experiments using the βS520C mutant ENaC which can be converted to a channel with a Po of nearly one, and by experiments using chymotrypsin to proteolytically activate the channel. These experiments revealed that the mutation reduced the average Po of ENaC by ñ75%. Na+ self inhibition of the mutant channel was significantly enhanced, but the observed effect was too small to account for the large reduction in average channel Po. The ENaC-activator S3969 partially rescued the loss-of-function phenotype of the αF61L mutation. We conclude that the αF61L mutation may contribute to respiratory symptoms in atypical CF patients.



 goto top of outline Author Contacts

Prof. Dr. med. Christoph Korbmacher
Institut für Zelluläre und Molekulare Physiologie
Waldstr. 6, D-91054 Erlangen (Germany)
Tel. +49 9131 8522301, Fax: +49 9131 8522770
E-Mail christoph.korbmacher@physiologie2.med.uni-erlangen.de


 goto top of outline Article Information

Accepted: September 08, 2009
Published online: December 22, 2009
Number of Print Pages : 14


 goto top of outline Publication Details

Cellular Physiology and Biochemistry (International Journal of Experimental Cellular Physiology, Biochemistry andPharmacology)

Vol. 25, No. 001, Year 2010 (Cover Date: 2010)

Journal Editor: F. Lang, Tübingen
ISSN: 1015–8987 (Print), eISSN: 1421–9778 (Online)

For additional information: http://www.karger.com/journals/cpb


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.