IKs response to protein kinase A-dependent KCNQ1 phosphorylation requires direct interaction with microtubules.
OBJECTIVE
KCNQ1 (alias KvLQT1 or Kv7.1) and KCNE1 (alias IsK or minK) co-assemble to form the voltage-activated K(+) channel responsible for I(Ks)-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.


RESULTS
Using a yeast two-hybrid screening, we detected a direct interaction between beta-tubulin and the KCNQ1 N-terminus. The interaction was confirmed by co-immunoprecipitation of beta-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 I(Ks). In addition, colchicine did not modify the current response to osmotic challenge. On the other hand, the I(Ks) 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.


CONCLUSIONS
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 I(Ks) activation.
Nicolas
Céline S
CS
Inserm, UMR915, l'institut du thorax, Nantes F-44035, France.
Park
Kyu-Ho
KH
El Harchi
Aziza
A
Camonis
Jacques
J
Kass
Robert S
RS
Escande
Denis
D
Mérot
Jean
J
Loussouarn
Gildas
G
Le Bouffant
Françoise
F
Baró
Isabelle
I
D054758:A Kinase Anchor Proteins / Q000378:metabolism; D000200:Action Potentials; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006168:Guinea Pigs; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D009997:Osmotic Pressure; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D050257:Tubulin Modulators / Q000494:pharmacology
D054758:A Kinase Anchor Proteins; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D014404:Tubulin; D050257:Tubulin Modulators; D017868:Cyclic AMP-Dependent Protein Kinases