Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations. Phosphatidylinositol (4,5)-bisphosphate (PIP2) is a phospholipid of the plasma membrane that has been shown to be a key regulator of several ion channels. Functional studies and more recently structural studies of Kir channels have revealed the major impact of PIP2 on the open state stabilization. A similar effect of PIP2 on the delayed rectifiers Kv7.1 and Kv11.1, two voltage-gated K+ channels, has been suggested, but the molecular mechanism remains elusive and nothing is known on PIP2 effect on other Kv such as those of the Shaker family. By combining giant-patch ionic and gating current recordings in COS-7 cells, and voltage-clamp fluorimetry in Xenopus oocytes, both heterologously expressing the voltage-dependent Shaker channel, we show that PIP2 exerts 1) a gain-of-function effect on the maximal current amplitude, consistent with a stabilization of the open state and 2) a loss-of-function effect by positive-shifting the activation voltage dependence, most likely through a direct effect on the voltage sensor movement, as illustrated by molecular dynamics simulations. Dual effect of phosphatidylinositol (4,5)-bisphosphate PIP2 on Shaker K+ channels.Journal of Biological ChemistryVol. 288Issue 15PreviewVOLUME 287 (2012) PAGES 36158–36167 Full-Text PDF Open Access Phosphatidylinositol (4,5)-bisphosphate (PIP2) 4The abbreviations used are: PIP2phosphatidylinositol (4,5)-bisphosphateTMRMtetramethylrhodamine-5-maleimidePMTphotomultiplier tubePMEparticle mesh EwaldMDmolecular dynamics. is a negatively charged phospholipid of the inner leaflet of the plasma membrane that is a key player in a variety of cellular processes. It has been demonstrated to be involved in the production of the second messengers inositol trisphosphate and diacylglycerol, in cytoskeletal organization, membrane trafficking, and regulation of ion channels and transporters activities (1Hilgemann D.W. Feng S. Nasuhoglu C. The complex and intriguing lives of PIP2 with ion channels and transporters.Sci. STKE. 2001; 2001: re19Crossref PubMed Scopus (486) Google Scholar, 2McLaughlin S. Wang J. Gambhir A. Murray D. PIP(2) and proteins: interactions, organization, and information flow.Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 151-175Crossref PubMed Scopus (694) Google Scholar). Examples of PIP2-dependent ion channels and receptors include inwardly rectifying K+ channels (Kir) (3Huang C.L. Feng S. Hilgemann D.W. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by G[β][γ].Nature. 1998; 391: 803-806Crossref PubMed Scopus (763) Google Scholar, 4Hilgemann D.W. Ball R. Regulation of cardiac Na+,Ca2+ exchange, and KATP potassium channels by PIP2.Science. 1996; 273: 956-959Crossref PubMed Scopus (558) Google Scholar), voltage-gated K+ channels (Kv) (5Zhang H. Craciun L.C. Mirshahi T. Rohács T. Lopes C.M. Jin T. Logothetis D.E. PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents.Neuron. 2003; 37: 963-975Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 6Park K.H. Piron J. Dahimene S. Mérot J. Baró I. Escande D. Loussouarn G. Impaired KCNQ1-KCNE1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome.Circ. Res. 2005; 96: 730-739Crossref PubMed Scopus (99) Google Scholar, 7Bian J. Cui J. McDonald T.V. HERG K(+) channel activity is regulated by changes in phosphatidylinositol 4,5-bisphosphate.Circ. Res. 2001; 89: 1168-1176Crossref PubMed Scopus (121) Google Scholar, 8Rodriguez N. Amarouch M.Y. Montnach J. Piron J. Labro A.J. Charpentier F. Mérot J. Baró I. Loussouarn G. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilizes the open pore conformation of the Kv11.1 (hERG) channel.Biophys. J. 2010; 99: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), voltage-gated Ca2+ channels (9Wu L. Bauer C.S. Zhen X.G. Xie C. Yang J. Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2.Nature. 2002; 419: 947-952Crossref PubMed Scopus (189) Google Scholar), TRP channels (10Rohács T. Lopes C.M. Michailidis I. Logothetis D.E. PI(4,5)P2 regulates the activation and desensitization of TRPM8 channels through the TRP domain.Nat. Neurosci. 2005; 8: 626-634Crossref PubMed Scopus (487) Google Scholar), and NMDA receptors (11Michailidis I.E. Helton T.D. Petrou V.I. Mirshahi T. Ehlers M.D. Logothetis D.E. Phosphatidylinositol-4,5-bisphosphate regulates NMDA receptor activity through α-actinin.J. Neurosci. 2007; 27: 5523-5532Crossref PubMed Scopus (44) Google Scholar, 12Mandal M. Yan Z. Phosphatidylinositol (4,5)-bisphosphate regulation of N-methyl-d-aspartate receptor channels in cortical neurons.Mol. Pharmacol. 2009; 76: 1349-1359Crossref PubMed Scopus (22) Google Scholar). phosphatidylinositol (4,5)-bisphosphate tetramethylrhodamine-5-maleimide photomultiplier tube particle mesh Ewald molecular dynamics. Functional studies (13Shyng S.L. Nichols C.G. Membrane phospholipid control of nucleotide sensitivity of KATP channels.Science. 1998; 282: 1138-1141Crossref PubMed Scopus (484) Google Scholar, 14Sui J.L. Petit-Jacques J. Logothetis D.E. Activation of the atrial KACh channel by the βγ subunits of G proteins or intracellular Na+ ions depends on the presence of phosphatidylinositol phosphates.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 1307-1312Crossref PubMed Scopus (213) Google Scholar) and more recently, structural studies on Kir channels (15Whorton M.R. MacKinnon R. Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium.Cell. 2011; 147: 199-208Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 16Hansen S.B. Tao X. MacKinnon R. Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2.Nature. 2011; 477: 495-498Crossref PubMed Scopus (436) Google Scholar) demonstrated that PIP2 acts by stabilizing the channels in an open state. Indirect evidence suggests that such open state stabilization by PIP2 might also be true for Kv channels, as indicated for Kv7.1 (17Loussouarn G. Park K.H. Bellocq C. Baró I. Charpentier F. Escande D. Phosphatidylinositol 4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels.EMBO J. 2003; 22: 5412-5421Crossref PubMed Scopus (173) Google Scholar) and Kv11.1 (8Rodriguez N. Amarouch M.Y. Montnach J. Piron J. Labro A.J. Charpentier F. Mérot J. Baró I. Loussouarn G. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilizes the open pore conformation of the Kv11.1 (hERG) channel.Biophys. J. 2010; 99: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The molecular mechanism underlying this PIP2 regulation of Kv channels is still unclear, although it was shown that mutations of positive residues in the cytosolic C terminus significantly reduce PIP2 affinity for those channels (6Park K.H. Piron J. Dahimene S. Mérot J. Baró I. Escande D. Loussouarn G. Impaired KCNQ1-KCNE1 and phosphatidylinositol-4,5-bisphosphate interaction underlies the long QT syndrome.Circ. Res. 2005; 96: 730-739Crossref PubMed Scopus (99) Google Scholar, 18Bian J.S. Kagan A. McDonald T.V. Molecular analysis of PIP2 regulation of HERG and IKr.Am. J. Physiol. Heart Circ. Physiol. 2004; 287: H2154-H2163Crossref PubMed Scopus (65) Google Scholar, 19Thomas A.M. Harmer S.C. Khambra T. Tinker A. Characterization of a binding site for anionic phospholipids on KCNQ1.J. Biol. Chem. 2011; 286: 2088-2100Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Interestingly, in Kv channels but also in voltage-gated Na+ channels (Nav), positive residues of the voltage sensor S4 are close to the inner leaflet of the cell plasma membrane, especially in the closed state (20Jogini V. Roux B. Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment.Biophys. J. 2007; 93: 3070-3082Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 21Pathak M.M. Yarov-Yarovoy V. Agarwal G. Roux B. Barth P. Kohout S. Tombola F. Isacoff E.Y. Closing in on the resting state of the Shaker K(+) channel.Neuron. 2007; 56: 124-140Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 22Delemotte L. Tarek M. Klein M.L. Amaral C. Treptow W. Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 6109-6114Crossref PubMed Scopus (133) Google Scholar, 23Payandeh J. Scheuer T. Zheng N. Catterall W.A. The crystal structure of a voltage-gated sodium channel.Nature. 2011; 475: 353-358Crossref PubMed Scopus (1084) Google Scholar). This raises the question whether or not PIP2 can also modify the activity of these channels by direct modulation of the voltage-sensing mechanism. To address this issue, we compared the effect of PIP2 on ionic and gating currents of Shaker channel (24Tempel B.L. Papazian D.M. Schwarz T.L. Jan Y.N. Jan L.Y. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.Science. 1987; 237: 770-775Crossref PubMed Scopus (488) Google Scholar), which was used as a model in this study, as it allows recording of large gating currents. We observed non-concomitant and opposite effects on current amplitude and channel voltage dependence, suggesting a dual effect of PIP2 on channel activity. Gating current measurement and voltage-clamp fluorimetry suggest that a direct effect of PIP2 on the voltage-sensor movement underlies the PIP2-induced modification of the channel voltage dependence, but not of the current amplitude, suggesting two binding sites. The COS-7 cell line, derived from the African green monkey kidney, was obtained from the American Type Culture Collection (CRL-1651, Rockville, MD) and cultured in DMEM supplemented with 10% serum and antibiotics (100 IU/ml penicillin and 100 mg/ml streptomycin), all from GIBCO, (Paisley, Scotland). Cells were transiently transfected with the plasmids using Fugene-6 (Roche Molecular Biochemical, Indianapolis, IN) according to the standard protocol recommended by the manufacturer. The WT Shaker (clone E, kind gift from Toshinori Hoshi), the N terminus-deleted Shaker Δ6–46 (Shaker-IR) in which fast inactivation is removed (25Hoshi T. Zagotta W.N. Aldrich R.W. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region.Neuron. 1991; 7: 547-556Abstract Full Text PDF PubMed Scopus (569) Google Scholar), and the non-conducting Shaker-W434F and Shaker-IR-W434F mutants were expressed in COS-7 cells using a pGW1 expression vector. The W434F mutation was used to permanently inactivate channels (26Perozo E. MacKinnon R. Bezanilla F. Stefani E. Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels.Neuron. 1993; 11: 353-358Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 27Yang Y. Yan Y. Sigworth F.J. How does the W434F mutation block current in Shaker potassium channels?.J. Gen. Physiol. 1997; 109: 779-789Crossref PubMed Scopus (155) Google Scholar) to record gating currents. The plasmid coding for the green fluorescent protein (pEGFP) used to identify transfected cells was purchased from Clontech (Palo Alto, CA). For giant-patch experiments on Shaker and Shaker-IR, a total of 1 μg of DNA was used. For giant-patch experiments on Shaker-W434F and Shaker-IR-W434F, a total of 8 μg of DNA was used. In both cases, 20% pEGFP combined with 80% of DNA of interest were used for transfection in a well of a 12-well plate. From 24 to 72 h after transfection, COS-7 cells were mounted on the stage of an inverted microscope and constantly superfused at a rate of ∼2 ml/min. Experiments were performed at room temperature (23 ± 2 °C). Acquisition and analysis were performed using pCLAMP 10.2 software (Molecular Devices). Electrodes were connected to an Axopatch 200A amplifier (Axon Instruments). For giant-patch experiments, the procedure described by Hilgemann (Hilgemann, 1989) was adapted to excise giant patches from COS-7 cells. Pipettes were pulled from borosilicate glass capillaries (glass type 8250, King Precision Glass, Claremont, CA) on a vertical puller (P30, Sutter Instruments, Novato, CA) and fire-polished using a microforge (MF-83, Narishige, Tokyo, Japan) to obtain tip diameters of ∼10 μm for patch pipettes and ∼20 μm for excision pipettes. The excision pipette, filled with the standard bath solution (see below), was connected to a 20-ml syringe to apply suction for excision. A microperfusion system allowed local application and rapid change of the different experimental solutions (28Loussouarn G. Baró I. Escande D. KCNQ1 K+ channel-mediated cardiac channelopathies.Methods Mol. Biol. 2006; 337: 167-183PubMed Google Scholar). In this giant-patch configuration, series resistances (around 0.5 MOhm) were not compensated, leading to a maximal error of 2 mV in the recordings with the current of the highest amplitude during the depolarizing pulse. Plasma membranes were held at −80 mV, depolarized to various potentials during 40 ms, from −10 to −75 mV, with 2.5 mV decrements, then repolarized to −80 mV, every 5 s. This protocol allowed several measurements: Maximal current, measured as the maximal activation current amplitudes at −10 mV obtained by fitting both activation and inactivation phases with Equation 1,I(t)=Imax×(1-exp(-t/τact))4×(r-(r-1) ×exp(-t/τinact))(Eq. 1) where Imax is the maximal current amplitude, τact the time constant of activation, τinact the time constant of inactivation, and r the residual percentage of current upon full inactivation. This equation allowed determining both activation and inactivation time constants at different potentials. Half-activation potential and slope factor of activation curve were obtained by fitting the maximal current at each step divided by the electromotive force, with a Boltzmann function (Equation 2),Irel=1/(1+exp(-(Vm-V1/2)/k))(Eq. 2) where Vm is the membrane potential, V1/2 is the half-activation potential, and k is the slope factor. Plasma membranes were held at −80 mV, depolarized to −10 mV during 50 ms, then repolarized to various potentials during 10 ms, from −20 to −110 mV, with 5-mV decrements, and finally repolarized to −80 mV, every 5 s. This protocol allowed determining the deactivation time constants at different potentials by fitting relaxation of ion current upon the first repolarization with a single exponential. Membrane holding potential was −80 mV. Plasma membranes were depolarized (pre-pulse) during 4 s to various potentials, from −95 to −15 mV, with 5 mV increments, then depolarized (pulse) to +50 mV during 250 ms, before being repolarized to −80 mV, every 5 s. Inactivation curves, obtained by normalizing maximum amplitude upon second pulse, were fitted with the following Boltzmann Equation 3,Irel=(1/1-exp(+(Vm-V1/2)/k))(Eq. 3) where Vm is the membrane potential, V1/2 is the half-activation potential, and k is the slope factor. This inactivation curve allowed characterizing of half-inactivation potential and slope factor of inactivation curve. Plasma membranes were held at −80 mV or −100 mV. An initial 200 ms pulse (prepulse) to +20 mV was followed by a second similar pulse (pulse) after an interval from 0 to 2 s, with 0.1 s increments. Pulse/prepulse relative amplitude was plotted against time interval. Recovery from inactivation time constants were obtained from the single exponential fit of recovery from inactivation curves. Activating ON gating currents (IgON) were elicited during 30 ms depolarizing potentials between −100 and +60 mV (with +10 mV increments) starting from a −80 mV holding potential. Subsequently, deactivating OFF gating currents (IgOFF) were recorded during a 30 ms repolarizing step to −100 mV. Capacitive and leak currents were subtracted using a -P/4 protocol starting from −100 mV or holding potential. Interpulse interval was 5 s long. This protocol allowed to determine: (i) the total amount of gating charges moved, measured by integrating the ON gating current at +60 mV and (ii) the voltage dependence of charge movement (half-activation potential and slope factor) by plotting the amount of gating charge as a function of depolarizing potential (Q-V curve) and fitting the relation with a Boltzmann function (Equation 2). Oocytes were prepared as previously reported (29Es-Salah-Lamoureux Z. Fougere R. Xiong P.Y. Robertson G.A. Fedida D. Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.PLoS ONE. 2010; 5: e10876Crossref PubMed Scopus (33) Google Scholar). Oocytes were placed in a bath chamber that was perfused with control ND96 bath solution containing (in mmol/liter), 96 NaCl, 3 KCl, 1 MgCl2, 2 CaCl2, and 5 HEPES, titrated to pH 7.4 with NaOH. Microelectrodes were filled with 3 mol/liter KCl and had resistances of 1 to 5 mΩ. Voltage control and data acquisition was achieved with a Warner Instruments OC-725C amplifier (Hamden, CT), and Axon Digidata 1322 A/D converter (Axon Instruments, Foster City, CA), connected to a personal computer running pClamp9 software (Molecular Devices Corp.). Fluorimetry was performed on the Shaker-IR-A359C-C445V (30Mannuzzu L.M. Moronne M.M. Isacoff E.Y. Direct physical measure of conformational rearrangement underlying potassium channel gating.Science. 1996; 271: 213-216Crossref PubMed Scopus (400) Google Scholar, 31Cha A. Bezanilla F. Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence.Neuron. 1997; 19: 1127-1140Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) simultaneously with two-electrode voltage clamp. Labeling of the oocytes with tetramethylrhodamine-5-maleimide (TMRM; Invitrogen, Carlsbad, CA) dye was performed at 10 °C in a depolarizing solution containing (in mmol/liter), 100 KCl, 1.5 MgCl2, 0.5 CaCl2, and 10 HEPES, titrated to pH 7.4 using KOH, with 5 μmol/liter TMRM. After 30 min of labeling, oocytes were stored in ND96 solution in the dark until voltage-clamped. Fluorimetry was performed using a Nikon TE300 inverted microscope with Epi-Fluorescence attachment and a 9124b Electron Tubes photomultiplier tube (PMT) module (Cairn Research, Kent, UK) as was described previously (Es-Salah-Lamoureux et al., 29Es-Salah-Lamoureux Z. Fougere R. Xiong P.Y. Robertson G.A. Fedida D. Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.PLoS ONE. 2010; 5: e10876Crossref PubMed Scopus (33) Google Scholar). To minimize fluorophore bleaching, a Uniblitz computer-controlled shutter (Vincent Associates, Ottawa, ON, Canada) was used, and opened shortly prior to application of voltage clamp pulses. Fluorescence signal sampling frequency was 6.67 kHz; signal traces were filtered offline at 300–1000 Hz. To correct for photobleaching of fluorophore that occurred during shutter opening during activation protocol and single sweep experiments, control fluorescence data were recorded in the absence of any change in voltage, and subtracted from the voltage-dependent signal. Membrane holding potential was −80 mV. Membranes were depolarized during 100 ms to various potentials, from −120 to 60 mV, with 10 mV increments, then repolarized to −80 mV, every 2 s. For giant-patch experiments, cells were superfused with a standard solution containing (in mm) 145 KCl, 10 HEPES, and 1 EGTA, pH 7.3 with KOH. A solution of (in mm) 145 K-gluconate, 10 HEPES, and 1 EGTA, pH 7.3 with KOH, was used to superfuse the cell during measurements and to fill the tip of the patch pipettes. Polylysine (Sigma-Aldrich) was diluted to 25 μg/ml before use. PIP2 (Calbiochem, Villeneuve d'Ascq, France) was diluted to 5 μm and sonicated on ice for 30 min before application to inside-out patches. We used all-atom models of the equilibrated open and closed conformations of the Kv1.2 embedded in fully hydrated POPC lipid bilayer (22Delemotte L. Tarek M. Klein M.L. Amaral C. Treptow W. Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 6109-6114Crossref PubMed Scopus (133) Google Scholar). The first ring of the bottom leaflet of POPC molecules around the Kv1.2 was replaced by PIP2 molecules (54 molecules for the closed conformation and 65 molecules for the open conformation) (supplemental Fig. S2). The system was then solvated in 150 mm KCl solution (A total of ∼350,000 atoms) and gradually relaxed using a standard procedure: the entire protein was fixed for 6 ns, enabling reorganization of the lipid and solution, then the backbone only for 2 ns, enabling relaxation of the side chains before finally letting the system relax freely for over 50 ns until reaching equilibrium. The MD simulations were carried out using the program NAMD2 (32Phillips J.C. Braun R. Wang W. Gumbart J. Tajkhorshid E. Villa E. Chipot C. Skeel R.D. Kalé L. Schulten K. Scalable molecular dynamics with NAMD.J. Comput. Chem. 2005; 26: 1781-1802Crossref PubMed Scopus (13144) Google Scholar). Langevin dynamics was applied to keep the temperature (300 K) fixed. The equations of motion were integrated using a multiple time-step algorithm. Short- and long-range forces were calculated every 1 and 2 time steps, respectively, with a time step of 2.0 fs. Chemical bonds between hydrogen and heavy atoms were constrained to their equilibrium value. Full three-dimensional periodic boundary conditions were used and long-range electrostatic forces were taken into account using the particle mesh Ewald (PME) approach. The water molecules were described using the TIP3P model (33Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. Comparison of simple potential functions for simulating liquid water.J. Chem. Phys. 1983; : 926-935Crossref Scopus (29878) Google Scholar). The simulation used the CHARMM22-CMAP force field with torsional cross-terms for the protein (34Mackerell Jr., A.D. Feig M. Brooks 3rd, C.L. Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations.J. Comput. Chem. 2004; 25: 1400-1415Crossref PubMed Scopus (2834) Google Scholar), CHARMM36 for the POPC phospholipids (35Klauda J.B. Venable R.M. Freites J.A. O'Connor J.W. Tobias D.J. Mondragon-Ramirez C. Vorobyov I. MacKerell Jr., A.D. Pastor R.W. Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types.J. Phys. Chem. B. 2010; 114: 7830-7843Crossref PubMed Scopus (2770) Google Scholar), and the CHARMM-compatible parameters for PIP2 developed in the group of Pr. Osman (36Lupyan D. Mezei M. Logothetis D.E. Osman R. A molecular dynamics investigation of lipid bilayer perturbation by PIP2.Biophys. J. 2010; 98: 240-247Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The simulations were performed on the SGI ALTIX ICE Machine JADE at the CINES supercomputer center (Montpellier, France). Statistical significance of the observed effects was assessed by Student's t test or two-way ANOVA test, using SigmaStat 3.1 software. p < 0.05 was considered significant. The effect of PIP2 on Shaker potassium channels was studied using the inside-out configuration of the patch-clamp technique. Patch excision of the membrane of a COS-7 cell expressing Shaker-IR mutant, in which the amino terminus responsible for the fast (N-type) inactivation was removed (25Hoshi T. Zagotta W.N. Aldrich R.W. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region.Neuron. 1991; 7: 547-556Abstract Full Text PDF PubMed Scopus (569) Google Scholar), led to a current rundown, potentially attributable to a decrease in membrane PIP2 levels. Consistent with that, addition on the inner side of the membrane of 25 μg/ml of a PIP2-scavenger, polylysine (Fig. 1, lower panel), accelerated the observed rundown, and eventually led to a complete loss of the channel activity (data not shown). Thus, polylysine was only transiently added (∼25 s) to reduce the current to approximately one-half of its initial value in order to maintain enough current to measure the biophysical parameters (Fig. 1, left lower panel). Polylysine removal did not lead to recovery of the current, suggesting that polylysine was not blocking the pore. Most importantly, intracellular addition of 5 μm PIP2 restored the current amplitude (Fig. 1, middle lower panel) confirming that the observed rundown is PIP2-dependent as previously reported for Kv11.1 and Kv7.1 channels (5Zhang H. Craciun L.C. Mirshahi T. Rohács T. Lopes C.M. Jin T. Logothetis D.E. PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents.Neuron. 2003; 37: 963-975Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 8Rodriguez N. Amarouch M.Y. Montnach J. Piron J. Labro A.J. Charpentier F. Mérot J. Baró I. Loussouarn G. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilizes the open pore conformation of the Kv11.1 (hERG) channel.Biophys. J. 2010; 99: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 17Loussouarn G. Park K.H. Bellocq C. Baró I. Charpentier F. Escande D. Phosphatidylinositol 4,5-bisphosphate, PIP2, controls KCNQ1/KCNE1 voltage-gated potassium channels: a functional homology between voltage-gated and inward rectifier K+ channels.EMBO J. 2003; 22: 5412-5421Crossref PubMed Scopus (173) Google Scholar). To fully characterize the effect of PIP2 on channel function, biophysical parameters were recorded at three distinct periods: just after patch excision (“ctrl”), after a 25 s application of polylysine (“ctrl post-poly-K”), and at steady state after ∼15 min (14.3 ± 3.3 min) of 5 μm PIP2 application (“PIP2”). Of note, since excision is associated with the dilution of many cytosolic components, the variation of the biophysical parameters observed between “ctrl” and “ctrl post-poly-K” may be due to other factors in addition to PIP2 decrease (8Rodriguez N. Amarouch M.Y. Montnach J. Piron J. Labro A.J. Charpentier F. Mérot J. Baró I. Loussouarn G. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilizes the open pore conformation of the Kv11.1 (hERG) channel.Biophys. J. 2010; 99: 1110-1118Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). For this reason, we rather focused on the comparison between “ctrl post-poly-K” and “PIP2” conditions. Ionic current recordings from a representative patch are shown in Fig. 1, upper panel. Membrane depolarization led to fast activation of the channels, followed by the expected remaining slow C-type inactivation (25Hoshi T. Zagotta W.N. Aldrich R.W. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region.Neuron. 1991; 7: 547-556Abstract Full Text PDF PubMed Scopus (569) Google Scholar). From the activation protocol (Fig. 1, inset) fitting both activation and inactivation with Equation 1 allowed determining accurately the maximal current at −10 mV in the three conditions (Fig. 1, right lower panel). The average maximal current, which is decreased after 25-s addition of polylysine, recovers to its original value upon addition of 5 μm PIP2, demonstrating a gain-of-function effect of the phospholipid on the current amplitude. To investigate the PIP2 effects on Shaker activation gating in greater detail, we studied the voltage-dependence of activation and the activation and deactivation kinetics (Fig. 2). Data from the activation protocol (Fig. 1, upper panel) were used to construct the activation curves and to determine the activation kinetics using Equation 1 (see “Experimental Procedures.”). After ∼15 min of PIP2 application, the activation curves were shifted by about +15 mV (Fig. 2, A and C), and the slope factor was slightly decreased (Fig. 2D). The decrease in the slope factor may be due to an incomplete decrease in PIP2 leading to a combination of channels with two profiles (PIP2-bound and PIP2-free), resulting in a shallower activation curve at a macroscopic level. In addition, PIP2 application significantly slowed the activation kinetics measured at potentials from −42.5 to −10 mV (Fig. 2B), and accelerated the deactivation kinetics at different potentials (Fig. 2, E and F). All the parameters (activation curve, activation, and deactivation kinetics) showed a similar 15-mV shift in voltage dependence (Fig. 2, A, B, and F), suggesting that the PIP2-induced loss-of-function might be acting on the closed to open equilibrium with no structural change in the activation mechanism. One could argue that open pore stabilization may interfere wit