HERG K+ Channel Activity Is Regulated by Changes in Phosphatidyl Inositol 4,5-Bisphosphate
Autonomic stimulation controls heart rate and myocardial excitability and may underlie the precipitation of both acquired and hereditary arrhythmias. Changes in phosphatidyl inositol bisphosphate (PIP2) concentration results from activation of several muscarinic and adrenergic receptors. We sought to investigate whether PIP2 changes could alter HERG K+ channel activity in a manner similar to that seen with inward rectifier channels. PIP2 (10 μmol/L) internally dialyzed increased the K+ current amplitude and shifted the voltage-dependence of activation in a hyperpolarizing direction. Elevated PIP2 accelerated activation and slowed inactivation kinetics. When 10 μmol/L PIP2 was applied to excised patches, no significant change in single channel conductance occurred, indicating that PIP2-dependent effects were primarily due to altered channel gating. PIP2 significantly attenuated the run-down of HERG channel activity that we normally observe after patch excision, suggesting that channel run-down is due, in part, to membrane depletion of PIP2. Inclusion of a neutralizing anti-PIP2 monoclonal antibody in whole cell pipette solution produced the opposite effects of PIP2. The physiological relevance of PIP2–HERG interactions is supported by our finding that phenylephrine reduced the K+ current density in cells coexpressing α1A-receptor and HERG. The effects of α-adrenergic stimulation, however, were prevented by excess PIP2 in internal solutions but not by internal Ca2+ buffering nor PKC inhibition, suggesting that the mechanism is due to G-protein–coupled receptor stimulation of PLC resulting in the consumption of endogenous PIP2. Thus, dynamic regulation of HERG K+ channels may be achieved via receptor-mediated changes in PIP2 concentrations.
Heart rate and contractility are continually altered in response to changing cardiovascular demands and stresses by means of autonomic stimulation. Some autonomic receptors (muscarinic M1 and α-adrenergic α1A) are coupled to Gαq proteins and, when activated, stimulate phosphatidyl inositol-specific phospholipase C (PLC). The substrate for PLC is phosphatidyl 4,5 bisphosphate (PIP2), a phospholipid that is hydrolyzed to 1,4,5 inositol trisphosphate (IP3) and diacyl glycerol (DAG). Thus, as these receptors are activated there is an increase in the second-messengers [IP3]i and [DAG]i with a simultaneous reduction of [PIP2] within the plasma membrane.
PIP2 itself plays an important role in such processes as the organization of actin cytoskeleton and vesicular transport,1,2 Na+/Ca2+ exchanger activity,3 IP3 receptor Ca2+ channel activity,4 and nonselective cation channels.5 PIP2 also regulates the activity of the ATP-sensitive potassium channel (KATP), a channel whose activity is gated by [ATP]i/[ADP]i changes.6–9 Other related K+ channels (GIRK) of the inward rectifier family are complexly regulated by the βγ-subunits of G-proteins and by simultaneous PLC-mediated changes in [PIP2].10–13
Because changes in autonomic stimulation of the heart appear to be linked to the precipitation of both hereditary and acquired ventricular arrhythmias,14 we decided to investigate whether changes in [PIP2] could affect HERG/IKr, one of the K+ channels that governs the rate of repolarization during the cardiac action potential. The human ether-a-go-go–related gene (HERG) encodes the pore-forming subunit of the channel that produces the cardiac rapidly activating delayed rectifier K+ current, Ikr, and mutations in HERG are responsible for the LQT2 form of hereditary long QT syndrome (LQTS). HERG/IKr is of the voltage-gated K+ channel family with a structure that is substantially different from those K+ channels that are known to be regulated by PIP2. No data, however, are available regarding the effect of [PIP2] changes on voltage-gated K+ channel activity.
Here, we report the effects of PIP2 on HERG channel activity. We show that modification of [PIP2] results in altered HERG/IKr current density and gating kinetics. Moreover, we show that stimulation of adrenergic α1A receptors affects coexpressed HERG channels in a manner expected for the PLC-mediated PIP2 consumption. These results support a model where autonomic stimulation of cardiac GPCRs may alter IKr-dependent repolarizing forces and thus, may be potentially arrhythmogenic.
Materials and Methods
Cell Culture and Transfection
CHO (from American Type Culture Collection, Manassas, Va) and HEK293 (a gift from Dr Craig January, Madison, Wis) cell lines were cultured in Ham’s F12 (Cellgro, CHO) and RPMI 1640 (Cellgro, HEK283) supplemented with l-glutamine, 10% fetal calf serum (BioWhittaker), and penicillin/streptomycin (Cellgro). Cultured cells were maintained in 5% CO2 humidified air at 37°C. For coexpression of HERG and GPCRs, 4 μg of α1A-adrenergic receptor cDNA was transiently cotransfected together with 4 μg of HERG cDNA and 2 μg of GFP cDNA by electroporation. Cells were electroporated in a 2-mm gap cuvette using a BTX ECM600 with a capacitance=180 μF; resistance=72 Ω; voltage=225 V. Cells were then plated sparsely on sterile glass cover and used for electrophysiological studies 24 to 72 hours after electroporation.
The entire coding region of adrenergic-α1A receptor cDNA (a generous gift from Dr Ken Minneman, Emory University) was subcloned into the EcoR I site pCDNA3 (Invitrogen). HERG and GFP expression plasmids have been previously described.15
Patch Clamp Recording
Patch pipettes were pulled and polished to obtain a tip resistance of 2 to 3 mΩ in the external bath solution. Fresh external bath solution was constantly flowed during all experiments to prevent accumulation of perimembrane K+ that would alter amplitude and kinetics of Ikr. All experiments were carried out at room temperature (20 to 22°C). Cells were studied on an inverted microscope equipped with electronic patch-pipette micromanipulators and epifluorescence optics. Axopatch-1D or 200B patch clamp amplifiers (Axon Instruments) were used for voltage clamp measurements. Voltage clamp protocols were controlled via PC using pClamp8 (Axon Instruments) acquisition and analysis software. To elicit HERG K+ currents depolarizing, voltage pulses were applied to various levels from a holding potential of −70 mV for 4.5 seconds followed by stepwise repolarization to −40 mV and then to −120 mV to measure outward and inward tail currents. To study the activation, inactivation, and deactivation of the HERG, channel-specific pulse protocols were used as described in the corresponding figure legends. Signals were analog-filtered at 2000 Hz and sampled at 5 to 10 000 Hz. Voltage-dependent activation data were fitted to Boltzmann relation I=1/(1+exp[(V1/2−V)/k]), where I is the measured tail current, V is the applied membrane voltage, V1/2 is the voltage at half-maximal activation, and k is the slope factor. To control for transfection efficiency, current densities were normalized to the control group daily based on average current density of cells before application of PIP2.
Whole-cell pipette solution consisted of (in mmol/L) KCl 126, MgSO4 2, CaCl2 0.5, EGTA 5, Mg-ATP 4, and HEPES 25 (pH 7.2; osmolality: 280±10 mmol/L). External bath solution consisted of (in mmol/L) NaCl 150, CaCl2 1.8, KCl 4, MgCl2 1, glucose 5, and HEPES 10 (pH 7.4; osmolality: 320±10 mmol/L). To buffer the increased [Ca2+]i during α1A-receptor stimulation, 15 mmol/L BAPTA was added to the pipette solution, and the [CaCl2]o was decreased to 0.5 mmol/L in the bath solution.
Single-channel recordings were performed in the inside-out and on-cell patch configuration using an Axopatch 200B amplifier (Axon Instruments). Single-channel data were digitally filtered at 500 or 600 Hz (low-pass) prior to analysis. Patch pipettes were made from thick-walled glass and coated with Sylgard to reduce noise and capacitance. Pipette solution for single channel recording consisted of (in mmol/L) KCl 120, MgCl2 1, CaCl2 1, and HEPES 25 (pH 7.4). Cells were bathed in a solution consisting of (in mmol/L) KCl 120, NaCl 10, MgCl2 2, glucose 5, and HEPES 10 (pH 7.4).
Cells were loaded with fluo-3 (Molecular Probes) at a final concentration of 2 μmol/L in RPMI medium. After incubation at 37°C for 45 minutes, the unincorporated dye was removed by washing the cells twice in the whole cell recording bath solution. The [Ca2+]i was measured in an inverted microscope (Nikon) that was outfitted with an excitation light source (excitation wavelength=480 nm). Fluorescent signals were obtained at 540 nm with a digital CCD camera (Quantix, Photometrics) controlled by commercial software (Axon Imaging Workbench 2.2, Axon Instruments). [Ca2+]i changes were expressed as fluorescence measured over basal unstimulated fluorescence (F/F0).
PIP2 and chelerythrine were from Calbiochem. To prepare PIP2, it was dispersed by sonication in water (at 0.5 mmol/L) for 30 minutes on ice and then divided into aliquots and keep at −80°C. For each experiment, a new aliquot was thawed and used only once. PIP2 was diluted to 10 μmol/L in the electrophysiology bath solution (for single-channel recording) or pipette solution (for whole-cell recording) and sonicated again for 10 to 30 minutes. This procedure results in the formation of a suspension of mostly small micelles of PIP2 that can be readily absorbed by lipid membranes.16 PIP2-specific antibody (Assay Designs Inc) was diluted to 60 nmol/L in the pipette solution.10,11 To decrease PIP2-Ab binding to the wall of the pipette, 100 μmol/L BSA was added to the pipette solution. Chelerythrine was first dissolved in DMSO as stock solutions and then used at the desired final concentration in the bath solution such that the final DMSO concentration was less than 0.5%. BAPTA was from Sigma.
Values presented are mean±standard error of mean (SEM). Student’s t test was used to compare the difference between 2 groups. Significance level was set at a value of P<0.05.
PIP2 Regulation of HERG K+ Channel Activity
We compared the HERG/IKr currents immediately after establishing whole cell and 3 minutes after allowing PIP2 (10 μmol/L) to dialyze into the cell (Figures 1A and 1B). We observed that 10 μmol/L PIP2 increased HERG/IKr current amplitude by 20% to 80%. The maximal current density as measured in I-V relationship increased from 3.58±0.83 to 4.72±1.05 pA/pF (P<0.01, n=15) (Figure 1C). Relative current amplitude differences were greatest between test voltages between −20 and +10 mV (PIP2-induced increase in current density 81% at −20 mV, 55% at −10 mV, 39% at 0 mV, and 20% at 10 mV). As shown in Figure 1D inset, when the data were normalized to unity, a 10 mV hyperpolarizing shift in voltage-dependent activation was seen (V1/2 HERG=−14.7±2.7 mV, V1/2 PIP2=−25.4±2.1 mV, P<0.05, n=15). The I-V relationship for the test pulse also showed a similar hyperpolarizing shift in voltage dependence of both the positive and negative slope conductances (Figure 1C inset, left). The PIP2 augmentation of HERG/IKr current was most pronounced at voltages negative to 0 mV (Figure 1C inset, right). The PIP2-dependent effects occurred rapidly and lasted at least 15 minutes (Figure 1E). There were no significant changes in membrane surface area (as measured by cell capacitance measurements) produced by PIP2 application to account for changes in current density.
PIP2 Acceleration of HERG K+ Channel Activation
We examined in detail the effect of increased [PIP2] on activation kinetics of HERG/IKr. PIP2 (10 μmol/L) produced an acceleration of the time constants at all membrane potentials tested (Figure 2A). The time constant for the outward current activation at −40 mV was accelerated by PIP2 from 2.34±0.47 to 1.67±0.22 seconds (P<0.05, n=8). As measured by rate of rise of outward current, this acceleration could be due to more rapid activation or slowing of voltage-dependent inactivation. To distinguish PIP2 effects on activation versus inactivation, we measured the time course of activation using a voltage clamp protocol varying the duration of the depolarizing pulse and measuring the peak inward tail current on repolarizing to −110 mV (Figures 2B and 2C). Because the inactivation of HERG channels is released at a faster rate than the channels deactivate,17 this is a more specific measure of activation. Figures 2B and 2C show the representative time course of the peak inward current on depolarization to different voltages (20 to 50 mV) in the control and PIP2 treatment group (Figures 2B and 2C). The half time to maximal rise in tail currents (T1/2) for both control and PIP2 treatment groups are summarized in Figure 2D. PIP2 significantly accelerated activation of current at all voltages measured.
PIP2 Effects on Inactivation and Deactivation of HERG
The amplitude of outward IKr and its rate of rise are determined by the relative degree of voltage-dependent activation and inactivation. Because both positive and negative slope conductances were shifted in response to increased [PIP2] (Figure 1C), voltage-dependent inactivation could be affected in addition to its effects on activation. To determine if [PIP2] changes altered inactivation kinetics, we used voltage clamp protocols to specifically measure voltage-dependence of steady-state inactivation in the procedure described by Smith et al.17 Our results show that increased [PIP2] significantly enhanced the current amplitude on release of inactivation (Figure 3A and 3B). Normalized data shows that there is a small but nonsignificant leftward shift in voltage-dependent inactivation (Figure 3B, inset). We also measured the rate of onset of inactivation before and after application of PIP2 (Figures 3C and 3D). Channels were activated by prolonged depolarization to +20 mV followed by a brief hyperpolarizing step to −120 mV and then depolarization to various potentials. The outward current reflects the open channel conductance prior to the onset of inactivation and onset of inactivation is seen in its declining phase to the steady state. Elevated [PIP2] significantly prolonged the time course of HERG inactivation, suggesting that PIP2 slows the onset of inactivation.
We examined whether elevation of [PIP2] exerted any effects on HERG/IKr deactivation using relaxation of tail current analyses (Figure 4A). The I-V relationship determined in this manner show the typical rectification with PIP2 producing increased amplitude, seen most prominently at potentials negative to −90 mV (Figure 4B). When relaxation time constants were examined from the tail currents, we observed no significant change in either the fast or slow rates of deactivation on application of [PIP2] (Figures 4C and 4D).
Effects of PIP2 on HERG/IKr in Excised Membrane Patches
To determine if the augmentation of whole cell current density in response to elevated [PIP2] was due to changes in single channel conductance, we examined HERG channels in excised membrane patches (Figure 5A). The slope conductance under these conditions was 10.7±2.3 pS (n=7) between the voltages of −40 and −120 mV in the absence of added PIP2. When the membrane patch was excised into solutions containing 10 μmol/L PIP2 the conductance was 10.2±0.8 pS (n=7), a value not significantly different from control (Figure 5B).
HERG channel activity in membrane patches consistently and quickly ran down after excision into the cell–free configuration (Figure 6A, upper traces). The run-down of the currents in excised patches has been reported for a wide variety of channels.10,18–24 Although the mechanisms for run-down are different for each channel and not entirely understood for all, one obvious explanation is the diffusion of necessary cytoplasmic cofactors on patch excision from the cell. We tested for loss of PIP2 as an explanation of HERG channel run-down. The integral of current amplitude and time was determined in multi-channel patches before and after excision to express the run-down of the channel activity. When 10 μmol/L PIP2 was included in the solution bathing the cytoplasmic surface of the patch there was less channel run-down (Figures 6A, lower traces, and 6B). Summary data for rates of channel run-down show a 5-fold reduction in loss of channel activity with PIP2 (Figure 6C). Loss of PIP2 through membrane associated phospholipases or diffusion from excised patches excision therefore, is at least partly responsible for the run-down of HERG channel activity and is consistent with PIP2 effects on channel rundown from other K+ channels.10–12
Effects of PIP2-Ab on the HERG Channel Activity
To test for the specificity of our observed PIP2 affects HERG/IKr, we used a neutralizing monoclonal PIP2-specific antibody that has been shown to disrupt PIP2 interactions with other K+ channel proteins.10,11,13 Inclusion of anti-PIP2 monoclonal antibody in whole cell pipette solution produced a significant reduction of HERG K+ current density (Figures 7A and 7B). Normalized data show a 7 mV depolarizing shift in voltage-dependent activation was seen (V1/2 HERG=−18.1±1.6 mV, V1/2 PIP2-Ab=−11±2.2 mV, n=7) (Figure 7B, inset). Because PIP2-Ab produced the inverse results that we obtained with elevated [PIP2], we conclude that the antibody bound to and neutralized the endogenous PIP2, thereby effecting a reduction of [PIP2] and its effects on HERG/IKr. Moreover, these results support the specificity of the PIP2 effects on the channel activity.
Effects of α1A-Adrenergic Receptor Stimulation on HERG K+ Current
To test whether physiological receptor-mediated alterations of [PIP2] could affect HERG/IKr, we cotransfected α1A-adrenergic receptor and HERG cDNAs into HEK293 cells. To verify that adequate receptor expression was achieved and whether they coupled to Gαq and PLC, we measured [Ca2+]i in response to receptor stimulation with phenylephrine. Receptor-mediated PLC activity will generate IP3, which releases [Ca2+]i from internal stores. The α1A-adrenergic agonist, phenylephrine (10 μmol/L), rapidly increased [Ca2+]i, demonstrating that the signaling pathway was intact (Figure 8A).
Activation of α1A-adrenergic receptor with 10-μmol/L phenylephrine resulted in a significant decrease in current amplitude and a small depolarizing shift in the voltage-dependence of activation (Figures 8B and 8C). The phenylephrine-dependent reduction in current occurred rapidly and was sustained (Figure 8B, inset). Activation of PLC generates the second messengers IP3 and DAG, which may alter channel behavior. To exclude the possibility that these effects are due to IP3-mediated [Ca2+]i elevation, BAPTA (15 mmol/L) was included in the whole cell pipette solution and the [Ca2+]o was reduced to 0.5 mmol/L. Under these conditions, we observed a comparable phenylephrine-mediated reduction in current amplitude (Figure 8E). To exclude the possibility of DAG-mediated activation of protein kinase, we treated cells with the PKC inhibitor chelerythrine (1 μmol/L) for 1 hour prior to patch clamp study. Similarly, under these conditions the phenylephrine-mediated reduction of HERG/IKr was intact (Figure 8F). As a further test for PIP2-specificity of the α-adrenergic effect, 10 μmol/L PIP2 internal was able to abolish current amplitude and voltage shifts from phenylephrine (Figure 8G). These data support the hypothesis that stimulation of G-protein–coupled receptors that activate PLC can alter endogenous [PIP2] sufficiently to modify the behavior of HERG K+ channels.
Polyvalent Cations Inhibited the PIP2 Effects on HERG K+ Channel
To investigate the possibility that the anionic head of PIP2 interacts with charged amino acids in HERG, we studied polyvalent cations ability to screen those charges. Although 100 μmol/L Gd3+ or La3+, when internally dialyzed, had no significant effect on HERG K+ current (data not shown), either could significantly inhibit the effect of PIP2 (Figure 8H). This is consistent with the previous studies of KATP channels.6,10,25
PIP2 has been shown to regulate the activity of several different K+ channels. Fan and Makiesk6 first described regulation of the KATP potassium channel by anionic phospholipids. Of the lipids tested, phosphatidyl inositol 4,5-bisphosphate (PIP2) was the most potent in augmenting channel activity. They identified positively charged (arginines) portions of the cytoplasmic N- and C-termini that appeared to be responsible for the lipid channel interaction and proposed that several other channels of the related inward rectifier class (ROMK, IRK, GIRK) may be similarly regulated.6 Nichols’s8 and Fakler’s7 groups then showed that PIP2 stimulated KATP channels by antagonizing the inhibitory effects of ATPi. Logothetis’s26 and Hilgemann’s10 groups showed that the G-protein–gated K+ channel (GIRK) required PIP2 for its normal activation by affecting the channel interactions with the βγ-G-protein subunits. Kobrinsky et al27 provided evidence that supports PIP2 as a dynamic physiological regulator of GIRK depending on rates of PIP2 hydrolysis by GPCR activation.
Although no voltage-gated K+ channels have been shown to interact with PIP2 to date, a suggestion of such an interaction is supported by the observation that LQTS arrhythmias are often precipitated by stress, presumably due to increased adrenergic stimulation of GPCRs in the heart. Heart rate and contractility are continually regulated by a multiplicity of hormonal and signaling information that impinges on the myocyte to control electrical properties required for different cardiovascular needs. β-Adrenergic–mediated increases in cAMP are the best studied so far of the HERG/IKr regulators,28–30 but other second messengers that are generated or altered via other receptors such as α-adrenergic or muscarinic invite further investigation. Our results show that increases in [PIP2] specifically lead to augmentation of HERG/IKr activity. This is accomplished by a combination of hyperpolarizing shift in voltage-dependent gating, accelerated activation, and slowed inactivation. Moreover, we show that activation of the α1A-adrenergic receptor, an activator of phosphatidyl inositol-specific PLC, leads to diminished HERG/IKr amplitude and a rightward shift in voltage dependence of activation as expected for PIP2 consumption.
The concentration of PIP2 (10 μmol/L) that we have used in this study is comparable to concentrations that have been used to demonstrate effects on KATP and inwardly rectifying K+ channels. Moreover, the amplitude of the electrophysiological response of HERG/IKr to PIP2 is likewise comparable to effects in these other K+ channels. Our demonstration that internal polyvalent cations could inhibit the PIP2 effects on HERG are supportive of electrostatic interactions between the oppositely charged potions of the channel and PIP2.6,9,10,31
The molecular mechanism of PIP2-dependent regulation of channels is postulated to be negatively charged phosphate groups of the PIP2 interacting with positively charged amino acids in the channel protein. The region of conserved cationic residues in the proximal C-terminal cytoplasmic domain of the inward rectifier K+ channels is considered to be a putative PIP2-binding site6 because charge-neutralizing mutations or deletion of this region markedly reduced the ability of channel to bind PIP2.10 Multiple potential PIP2 binding sites in Kir channels have now been proposed,25 not only in the proximal C-terminal but also in other C-terminal fragments. Three independent sites (aa175-206, aa207-246, aa324-365) that could potentially bind PIP2 were identified in the C-terminal tail of Kir2.1 channels.25 Several polycationic segments exist in the cytoplasmic C-terminus of HERG (611: RYHTQMLRVREFIRFHQIPNPLRQRL637 and 881: RKRKLSFRRRT895). Although there is no sequence homology between these segments and those documented in K-ATP, ROMK1, and GIRK channels, there is a consistent clustering of 5 to 7 positively charged amino acids (arginines and lysines) within a small region of the C-terminal cytoplasmic tail. Our demonstration that internal polyvalent cations could inhibit the PIP2 effects on HERG is supportive of electrostatic interactions between the oppositely charged potions of the channel and PIP2. Further biochemical studies are needed to determine the exact binding site of PIP2 to the HERG channel.
Some Kir channels have additional regulators that influence their dependence on PIP2. Kir1.1 (ROMK1) channels are regulated by protein kinase A (PKA) through phosphorylation that seems to enhance the interaction of these channels with PIP2.11 However, in the HERG K+ channel, PKA may not be involved in the interaction of PIP2 and HERG channel, because we previously found that phosphorylation of HERG channel by PKA does not increase but decreases HERG K+ current.29 This is complicated, however, by the complex regulation of HERG/IKr by cAMP due to the four PKA consensus sites and cyclic nucleotide-binding domain of HERG.29,30 Further investigation is needed to explore signaling cross talk between cAMP, PIP2, and HERG.
There is evidence that activation of PLC-linked GPCRs significantly alters cell membrane concentrations of PIP2. Willars et al32 showed that stimulation of the muscarinic M3 receptor reduced the basal PIP2 concentration to 15% of basal levels in a rapid and sustained manner. Moreover, they showed that the pool of receptor-sensitive PIP2 turned over every 5 seconds during sustained stimulation. That these receptor-mediated changes in [PIP2] can affect K+ channel function is supported by reports using KATP and GIRK channels.7,27 The effects of short- versus long-term stimulation of receptors and stimulation of receptors in cardiac myocytes will require further investigation.
The heart is presented with continually varying cardiovascular demands that require dynamic responses, both inotropic and chronotropic. Much of the cardiac adaptation that occurs is the result of changes in autonomic/hormonal stimulation involving G-protein–coupled receptors. Under normal situations these adaptations are harmless and meet the needs of the situation at hand; however, under other circumstances they can lead to maladaptive results including ventricular arrhythmias. The results in this study show, for the first time, that PIP2 regulates the activity of the voltage-gated K+ channel, HERG/IKr. These results provide an additional link between cardiovascular stresses, autonomic stimulation, and arrhythmias, both hereditary and acquired.
This work was supported by grants from the NIH/NHLBI (RO1 HS57388 to T.V.M.) and the American Heart Association (NYC Affiliate 0120335T to J.B.). We thank Yonathan Melman and Anna Kagan for critical reading of the manuscript.
Original received July 24, 2001; revision received October 25, 2001; accepted October 25, 2001.
Janmey PA, Xian W, Flanagan LA. Controlling cytoskeleton structure by phosphoinositide-protein interactions: phosphoinositide binding protein domains and effects of lipid packing. Chem Phys Lipids. 1999; 101: 93–107.
Raucher D, Stauffer T, Chen W, Shen K, Guo S, York JD, Sheetz MP, Meyer T. Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell. 2000; 100: 221–228.
Hilgemann DW, Ball R. Regulation of cardiac Na+,Ca2+ exchange and KATP potassium channels by PIP2. Science. 1996; 273: 956–959.
Lupu VD, Kaznacheyeva E, Krishna UM, Falck JR, Bezprozvanny I. Functional coupling of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1998; 273: 14067–14070.
Zhainazarov AB, Ache BW. Effects of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate on a Na+-gated nonselective cation channel. J Neurosci. 1999; 19: 2929–2937.
Fan Z, Makielski JC. Anionic phospholipids activate ATP-sensitive potassium channels. J Biol Chem. 1997; 272: 5388–5395.
Baukrowitz T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP, Fakler B. PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science. 1998; 282: 1141–1144.
Shyng SL, Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science. 1998; 282: 1138–1141.
Xie LH, Takano M, Kakei M, Okamura M, Noma A, Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels. J Physiol. 1999; 514: 655–665.
Huang CL, Feng S, Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ. Nature. 1998; 391: 803–806.
Liou HH, Zhou SS, Huang CL. Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. Proc Natl Acad Sci U S A. 1999; 96: 5820–5825.
Rohacs T, Chen J, Prestwich GD, Logothetis DE. Distinct specificities of inwardly rectifying K+ channels for phosphoinositides. J Biol Chem. 1999; 274: 36065–36072.
Zhang H, He C, Yan X, Mirshahi T, Logothetis DE. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat Cell Biol. 1999; 1: 183–188.
Roden DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, Vincent GM. Multiple mechanisms in the Long-QT Syndrome: current knowledge, gaps, and future directions. Circ Res. 1996; 94: 1996–2012.
McDonald TV, Yu Z, Ming Z, Palma E, Meyers MB, Goldstein SAN, Fishman GI. A minK-HERG complex regulates the cardiac potassium current IKr. Nature. 1997; 388: 289–292.
Flanagan LA, Cunningham CC, Chen J, Prestwich GD, Kosik KS, Janmey PA. The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and τ. Biophys J. 1997; 73: 1440–1447.
Smith PL, Baukrowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature. 1996; 379: 833–836.
Marty A, Neher E. Potassium channels in cultured bovine adrenal chromaffin cells. J Physiol. 1985; 367: 117–141.
Belles B, Malecot CO, Hescheler J, Trautwein W. “Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium. Pflugers Arch. 1988; 411: 353–360.
Stelzer A, Kay AR, Wong RK. GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science. 1988; 241: 339–341.
Wang WH. Two types of K+ channel in thick ascending limb of rat kidney. Am J Physiol. 1994; 267: F599–F605.
Fakler B, Brandle U, Glowatzki E, Zenner HP, Ruppersberg JP. Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis. Neuron. 1994; 13: 1413–1420.
Robertson GA, Warmke JM, Ganetzky B. Potassium currents expressed from Drosophila and mouse eag cDNAs in Xenopus oocytes. Neuropharmacology. 1996; 35: 841–850.
Costantin JL, Qin N, Waxham MN, Birnbaumer L, Stefani E. Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes: partial reversal by protein kinase A. Pflugers Arch. 1999; 437: 888–894.
Soom M, Schonherr R, Kubo Y, Kirsch C, Klinger R, Heinemann SH. Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels. FEBS Lett. 2001; 490: 49–53.
Sui JL, Petit-Jacques J, Logothetis DE. 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: 1307–1312.
Kobrinsky E, Mirshahi T, Zhang H, Jin T, Logothetis DE. Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization. Nat Cell Biol. 2000; 2: 507–514.
Thomas D, Zhang W, Karle CA, Kathofer S, Schols W, Kubler W, Kiehn J. Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A. J Biol Chem. 1999; 274: 27457–27462.
Cui J, Melman Y, Palma E, Fishman GI, McDonald TV. Cyclic AMP regulates the HERG K+ channel by dual pathways. Curr Biol. 2000; 10: 671–674.
Cui J, Kagan A, Qin D, Mathew J, Melman YF, McDonald TV. Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with kcne2. J Biol Chem. 2001; 276: 17244–17251.
Bokvist K, Olsen HL, Hoy M, Gotfredsen CF, Holmes WF, Buschard K, Rorsman P, Gromada J. Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells. Pflugers Arch. 1999; 438: 428–436.
Willars GB, Nahorski SR, Challiss RA. Differential regulation of muscarinic acetylcholine receptor-sensitive polyphosphoinositide pools and consequences for signaling in human neuroblastoma cells. J Biol Chem. 1998; 273: 5037–5046.