Cardiovascular Research Advance Access first published online on April 5, 2008
This version [Corrected Proof] published online on April 25, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn085
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IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules
1 Inserm, UMR915, l'institut du thorax, Nantes F-44035, France
2 CNRS, ERL3147, Nantes F-44035, France
3 Faculté de Médecine, Université de Nantes, Nantes F-44035, France
4 Inserm, U830, Institut Curie, Paris F-75248, France
5 Department of Pharmacology, Columbia University Medical Center, 630 West, 168th Street, New York, NY 10032, USA
* Corresponding author. Tel: +33 2 40 41 28 48; fax: +33 2 40 41 29 50. E-mail address: isabelle.baro{at}nantes.inserm.fr
Aims: KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K+ channel responsible for IKs—a major repolarizing current in the human heart—and their dysfunction promotes cardiac arrhythmias. The channel is a component of larger macromolecular complexes containing known and undefined regulatory proteins. Thus, identification of proteins that modulate its biosynthesis, localization, activity, and/or degradation is of great interest from both a physiological and pathological point of view.
Methods and results: Using a yeast two-hybrid screening, we detected a direct interaction between β-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of β-tubulin and KCNQ1 in transfected COS-7 cells and in guinea pig cardiomyocytes. Using immunocytochemistry, we also found that they co-localized in cardiomyocytes. We tested the effects of microtubule-disrupting and -stabilizing agents (colchicine and taxol, respectively) on the KCNQ1–KCNE1 channel activity in COS-7 cells by means of the permeabilized-patch configuration of the patch-clamp technique. None of these agents altered IKs. In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the IKs response to protein kinase A (PKA)-mediated stimulation depended on microtubule polymerization in COS-7 cells and in cardiomyocytes. Strikingly, KCNQ1 channel and Yotiao phosphorylation by PKA—detected by phospho-specific antibodies—was maintained, as was the association of the two partners.
Conclusion: We propose that the KCNQ1–KCNE1 channel directly interacts with microtubules and that this interaction plays a major role in coupling PKA-dependent phosphorylation of KCNQ1 with IKs activation.
KEYWORDS K+ channel; Myocytes; PKA; Signal transduction; KCNQ1; β-tubulin
Time for primary review: 31 days
Present address. IBBMC, Bâtiment 430, Université de Paris-Sud, F-91405 Orsay, France.
Present address. Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
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