Aims
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.
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 I(Ks) activation.
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.