Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP2 and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch–clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP2 and fully prevented by application of PIP2 plus MgATP. PIP2‐dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP‐dependent rundown was not. Cytosolic application of PIP2 slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP2 was fully restituted by a model originally elaborated for ATP‐regulated two transmembrane‐domain potassium channels. The model is consistent with stabilization by PIP2 of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane‐domain voltage‐gated channel and a two transmembrane‐domain ATP‐gated channel.

Nearly a hundred different KCNQ1 mutations have been reported as leading to the cardiac long QT syndrome, characterized by prolonged QT interval, syncopes, and sudden death. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) regulates the KCNQ1–KCNE1 complex. In the present study, we show that PIP₂ affinity is reduced in three KCNQ1 mutant channels (R243H, R539W, and R555C) associated with the long QT syndrome. In giant excised patches, direct application of PIP₂ on the cytoplasmic face of the three mutant channels counterbalances the loss of function. Reintroduction of a positive charge by application of methanethiosulfonate ethylammonium on the cytoplasmic face of R555C mutant channels also restores channel activity. The channel affinity for a soluble analog of PIP₂ is decreased in the three mutant channels. By using a model that describes the KCNQ1–KCNE1 channel behavior and by fitting the relationship between the kinetics of deactivation and the current amplitude obtained in whole-cell experiments, we estimated the PIP₂ binding and dissociation rates on wild-type and mutant channels. The dissociation rate of the three mutants was higher than for the wild-type channel, suggesting a decreased affinity for PIP₂. PIP₂ binding was magnesium-dependent, and the PIP₂-dependent equilibrium constant in the absence of magnesium was higher with the wild-type than with the mutant channels. Altogether, our data suggest that a reduced PIP₂ affinity of KCNQ1 mutants can lead to the long QT syndrome.

Hemifluorinated and fluorinated surfactants are lipophobic and, as such, non-detergent. Although they do not solubilize biological membranes, they can, after conventional solubilization, substitute for detergents to keep membrane proteins soluble, which generally improves their stability [Breyton, Chabaud, Chaudier, Pucci and Popot (2004) FEBS Lett. 564, 312–318]. In the present study, we show that (hemi)fluorinated surfactants can be used for in vitro synthesis of membrane proteins: they do not interfere with protein synthesis, and they provide a suitable environment for MscL, a pentameric mechanosensitive channel, to fold and oligomerize to its native functional state. Following synthesis, both types of surfactants can be used to deliver MscL directly to pre-formed lipid vesicles. The electrophysiological activity of MscL synthesized in vitro in the presence of either hemi- or per-fluorinated surfactant is similar to that of the protein expressed in vivo.

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. 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 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. 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.