title: Synergistic modulation of KCNQ1/KCNE1 K(+) channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP]i.
abstract: Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion. Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.
authors.affiliation: Institute of Physiology, Ruhr-University Bochum, D-44780 Bochum, Germany. Electronic address: cecile.kienitz@rub.de.
authors.forename: Marie-Cécile
authors.initials: MC
authors.lastname: Kienitz
authors.affiliation: Institute of Physiology, Ruhr-University Bochum, D-44780 Bochum, Germany.
authors.forename: Dilyana
authors.initials: D
authors.lastname: Vladimirova
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004357:Drug Synergism; D031541:Fluorescence Resonance Energy Transfer; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; D019269:Phosphatidylinositol 4,5-Diphosphate; D024642:Potassium Channels, Voltage-Gated; D043587:Receptor, Muscarinic M3; D000255:Adenosine Triphosphate; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000109:Acetylcholine
keywords: ATP; Electrophysiology; FRET; KCNQ1; Phosphatidylinositol