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Am J Physiol Heart Circ Physiol 287: H2154-H2163, 2004. First published July 1, 2004; doi:10.1152/ajpheart.00120.2004
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Molecular analysis of PIP2 regulation of HERG and IKr

Jin-Song Bian,1 Anna Kagan,2 and Thomas V. McDonald2

1Department of Pharmacology, Faculty of Medicine, National University of Singapore, 117597 Singapore; and 2Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

Submitted 5 February 2004 ; accepted in final form 28 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We previously reported that cloned human ether-à-go-go-related gene (HERG) K+ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP2) concentration. Here we investigated the molecular determinants of PIP2 interactions with HERG channel protein. To establish the molecular nature of the PIP2-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883–894) as a possible site of interaction with negatively charged PIP2. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP2, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP2-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP2 effects on channel-inactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of {alpha}1A-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K+ current (the current carried by ERG protein) from rabbit ventricular myocytes. The {alpha}-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP2 consumption that alters channel activity. These results support a physiological role for PIP2 regulation of the rapidly activating delayed rectifier K+ current during autonomic stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K+ channel.

human ether-à-go-go-related gene; phosphatidylinositol 4,5-bisphosphate; delayed rectifier K+ current; channel; phospholipids; G protein-coupled receptor; mutagenesis; phospholipase C

REGULATION OF ION CHANNEL function plays a pivotal role in the control of heart rate and contractility via changes in cardiac myocyte excitability. Humoral mediators and receptors are involved in determining channel responses to changing cardiovascular demands. The dynamic beat-to-beat regulation of ion channels is precisely controlled by autonomic stimulation through complex interplay of second messengers, kinases, G proteins, and protein-protein interactions. The rapidly activating delayed rectifier K+ current (IKr) is essential for proper repolarization of the cardiac myocyte at the end of each action potential (15, 23). Decreased abundance or malfunction of IKr increases the propensity to ventricular tachyarrhythmia (32). The gene that encodes the pore-forming subunit of the IKr channel is human ether-à-go-go-related gene (HERG), which has been linked to both hereditary and acquired ventricular arrhythmias (8, 19, 22, 29). Adrenergic stimulation has numerous effects on HERG, and IKr and has been best studied for {beta}-adrenergic receptor pathways (6, 7, 16, 30). In this study, we sought to investigate the {alpha}-adrenergic affects on HERG and IKr to better understand the complex control of this current during physiological stresses. A signaling pathway that is set in motion upon {alpha}-adrenergic stimulation involved G protein-mediated activation of PLC. Activated PLC generates diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3) as by-products from the consumption of the endogenous phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Changes in PIP2 concentration ([PIP2]) have been shown to regulate a variety of K+ channels (3, 9, 14, 17, 21, 26, 33, 34). Heterologously expressed HERG was the first voltage-gated channel shown to be responsive to changes in [PIP2] (4). The other LQT-linked delayed rectifier K+ current, IKs, which is composed of the KCNQ1 channel (with the KCNE1 subunit), has also been shown to be regulated in cardiac myocytes and in a heterologous expression system (18). We now show that native IKr in cardiac myocytes is responsive to PIP2 changes. Moreover, we have employed mutagenesis to map a site within the channel protein that binds to PIP2 and is responsible for much of the PIP2 responsiveness of HERG and IKr.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Construction of HERG COOH-terminal deletion. HERG-wild-type (WT)- and green fluorescent protein (GFP)-expression plasmids have been previously described (4, 7). Deletion or mutation of a portion of the HERG COOH terminus (amino acids 883–894) was constructed using a PCR-based approach. Briefly, using PCR with Pfu Turbo (Stratagene) and 5'-phosphorylated primers, we produced two blunt-ended PCR fragments. The forward primer for the first fragment begins at base pair 1608 (bp1608) of HERG cDNA (5'-TGGTGCGCGTGGCGCGGAAGCTG), and the reverse primers of D-HERG and M-HERG are from bp2659 (ACTGAAGCCACCCTCTAACTC) and bp2680 (CATCTTGATCTCGTCTGGCCGACTGAAGCCACC). The forward primers for the second fragment begin at bp2696 and are p-ACGGACAAGGACACGGAGC for deletion-HERG (D-HERG) and p-TCCTTCCCGAACGGCACGGACAAGGACACGGA for methionine-substituted-HERG (M-HERG). The reverse primer is CTCCACGTCGCCCCGGGGCCGCCGACC and starts at bp3130. The two blunt-ended fragments were gel purified and ligated overnight and reamplified by PCR for subcloning into PCR Blunt II TOPO (Invitrogen). The TOPO plasmid cDNAs containing the PCR products were digested with FseI and XhoI. The ~1,500-bp fragment was purified and ligated into the FseI-XhoI sites within HERG in HERG-myc-pCI-Neo. The final plasmid encoded a myc-tagged mutant HERG protein (for both D- and M-HERG).

Cell culture and transfection. Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cell lines (from American Type Culture Collection) were cultured in Ham's F-12 or RPMI 1640, respectively, and supplemented with L-glutamine, 10% fetal calf serum (Hyclone), and penicillin-streptomycin (GIBCO). Cultured cells were maintained in 5% CO2-95% humidified air at 37°C. We transiently cotransfected 5 µg of WT-HERG, D-HERG, or M-HERG cDNA to CHO cells together with 2 µg of GFP cDNA via electroporation. Cells were washed with cytomix buffer (in mM: 120 KCl, 0.15 CaCl2, 10 K2HPO4-KH2PO4, 25 HEPES, 2 EGTA, and 5 MgCl2, pH 7.6) and centrifuged at 300 g for 5 min. Cell suspension (400 µl) in complete cytomix buffer that contained 2 mM ATP and 5 mM glutathione was electroporated in a 2-mm gap cuvette using a BTX ECM600 electroporation system with the following settings: capacitance, 180 µF; resistance, 72 {Omega}; and voltage, 225 V. After electroporation, the cells were plated sparsely and grown on sterile glass coverslips in 100-mm tissue culture dishes. Cells were used for electrophysiological studies 24–72 h after electroporation. For biochemical analyses, transient transfections were performed with Lipofectamine 2000 (Invitrogen) as previously described (6, 16).

Isolation of rabbit cardiac myocytes. Male rabbits (1–2 kg) were anesthetized with an injection of 55 mg/kg im ketamine and 7 mg/kg im xylazine. Heparin (1,000 IU iv) was administered to prevent coagulation during heart removal. The heart was quickly excised, mounted on a Langendorff apparatus, and perfused in a retrograde fashion via the aorta with nominally Ca2+-free Tyrode solution at 37°C. The Tyrode solution contained (in mM) 137 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. After 5 min, the perfusion solution was changed to the Tyrode solution that contained 1 mg/ml type I collagenase (Sigma) and 0.28 mg/ml type XIV protease (Sigma) and was perfused for 25–30 min. The perfusion solution was then changed to Ca2+-Tyrode solution that contained 0.2 mM CaCl2 without proteases for 5 min. The ventricular tissue was then cut into small pieces in a petri dish with 20 ml of prewarmed Ca2+-Tyrode solution and was shaken gently inside the solution for the dispersion of dissociated cardiac myocytes. A 250-µM mesh screen was used to separate the isolated cardiac myocytes from cardiac tissue. The cells were washed three times in Ca2+-Tyrode solution and collected by centrifugation at 100 rpm for 1 min. Isolated myocytes were resuspended in Ca2+-Tyrode solution and plated onto laminin (Sigma)-coated glass coverslips. Cells were allowed to attach for 2 h and then were used for electrophysiology studies.

Patch-clamp recordings. Cells on coverslips were taken directly from the cell culture incubator and placed in an acrylic-polystyrene perfusion chamber (Warner Instruments) for electrophysiological measurements. For the rabbit cardiac myocytes, rod-shaped myocytes with clear striations were selected for electrical recordings. Patch pipettes were pulled and polished to obtain a tip resistance of 2–3 M{Omega} in the patch-clamp solutions. Pipette offset potential in our study solutions was compensated to zero just before gigaseal formation. The junction potential in the experimental solutions was estimated to be 3–4 mV (by pCLAMP 9 analysis software) and was not corrected for analysis. Whole cell capacitance was compensated electronically through the amplifier. The series resistance in the whole cell configuration was between 9 and 10 M{Omega}. All experiments were carried out at room temperature (20–22°C). Cells were studied on an inverted microscope equipped with electronic patch-pipette micromanipulators and epifluorescence optics for GFP (transfected cells). Axopatch 200B patch-clamp amplifiers (Axon Instruments) were used for voltage-clamp measurements. Voltage-clamp protocols were controlled via personal computer using pCLAMP 9 acquisition and analysis software. To elicit HERG K+ currents, depolarizing voltage pulses were applied at various levels from a holding potential of –70 mV for 4.5 s and were followed by stepwise repolarization to –40 mV and then to –120 mV to measure outward and inward tail currents, respectively. Signals were analog filtered at 2,000 Hz and sampled at 5–10,000 Hz. Voltage-dependent activation data were fitted to the Boltzmann relation I = 1/{1 + exp[(Vh – V)/k]}, where I is the relative tail current amplitude, V is the applied membrane voltage, Vh is the voltage at half-maximal activation, and k is the slope factor (22). To compare the effects before and after the administration of reagents, current amplitude was normalized to the control group before application of drugs.

For whole cell voltage-clamp measurements, the pipette solution consisted of (in mmol/l) 126 KCl, 2 MgSO4, 0.5 CaCl2, 5 EGTA, 4 Mg-ATP, and 25 HEPES (pH 7.2; osmolality, 280 ± 10 mosmol/kg). The external bath solution consisted of (in mM) 150 NaCl, 1.8 CaCl2, 4 KCl, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4; osmolality, 320 ± 10 mosmol/kg). To increase the cellular [PIP2] directly, 10 µM PIP2 was included in the pipette solution. For IKr recordings in the cardiac myocytes, the extracellular solution contained (in mM) 150 N-methyl-D-glucamine (NMDG), 1.8 CaCl2, 4 KCl, 1 MgCl2, 5 glucose, and 10 HEPES (pH 7.4; osmolality, 320 ± 10 mosmol/kg) at room temperature (1). To buffer the increased intracellular Ca2+ concentration ([Ca2+]i) upon {alpha}1A-receptor stimulation, 15 mM BAPTA was added to the pipette solution (with appropriate pH adjustment).

In vitro PIP2 binding assay. HEK-293 cells were transfected with WT-HERG-myc, D-HERG-myc, M-HERG-myc, or Kir2.1-myc. At 48 h after transfection, cells were washed with ice-cold PBS and lysed in ice-cold buffer that contained 150 mM NaCl, 25 mM Tris·HCl, pH 7.5, 5 mM EDTA, 1% Nonidet P-40, 0.4% deoxycholic acid, and EDTA-free protease inhibitor cocktail tablets (Roche Pharmaceuticals). Lysates were cleared by centrifugation at 13,200 rpm for 10 min at 4°C. The supernatant was incubated with 20 µl of rabbit polyclonal A14 anti-myc antibody (Santa Cruz) and 60 µl of protein G-agarose (Pierce) for 4 h at 4°C. Precipitated proteins were washed with 0.25% Nonidet P-40 buffer that contained 150 mM NaCl, 25 mM Tris·HCl, pH 7.5, and 0.25% Nonidet P-40 and were then washed with PBS. A 1:1 mixture of [3H]PIP2 in chloroform-methanol-buffered saline (sp. act., 8 µCi/nmol; American Radiolabeled Chemicals) was dried by vacuum at 4°C and sonicated in 100 µl of PBS to form pure [3H]PIP2 liposomes. The [3H]PIP2 liposomes were incubated with the immobilized proteins for 2 h at 4°C with or without varying concentrations of unlabeled PIP2. After precipitates were washed twice with PBS, 10% were dissolved in SDS gel-loading buffer, and 90% of the proteins were counted in a scintillation counter (Beckman) using a window for 3H. The SDS-PAGE was probed with mouse monoclonal 9E10 anti-myc antibody and subjected to immunoblot analysis. Specific binding was obtained by subtracting the background binding value obtained from protein G antibody applied to samples from nontransfected HEK-293 cells.

To determine PIP2 affinity, different concentrations of unlabeled PIP2 (10–7 to 10–3 M) were used to compete with 0.5 µM [3H]PIP2 for binding. The competition data were analyzed using the equation (binding of radiolabeled ligand) = Bmax([3H]PIP2)/([3H]PIP2) + [(unlabeled PIP2) + Kd].

Materials. PIP2, bisindolymaleimide I (BSM), and chelerythrine were purchased from Calbiochem. Collagenase type I, protease type XIV, laminin, and BAPTA were from Sigma. Chromanol 293B was obtained from Tocris. For preparation of PIP2, the lipid was first dispersed in water (at 0.5 mM concentration) by sonication for 30 min on ice and then divided into aliquots and kept at –80°C. For each experiment, a new aliquot was thawed and used only once. PIP2 was diluted to 10 µM in the electrophysiology pipette solution and sonicated again for 10–30 min. This procedure results in the formation of a suspension of mostly small micelles of PIP2 that can be readily absorbed by lipid membranes (10). Chelerythrine and BSM were first dissolved in DMSO as stock solutions and then used at the desired final concentrations in the bath solution such that the final DMSO concentration was <0.5%.

Statistics. Values presented are means ± SE. Student's t-test was used to compare the differences between two groups. Significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
D-HERG and M-HERG attenuate effects of PIP2. To determine whether the charged segment (amino acids 883–894) in the cytoplasmic COOH-terminal tail of HERG is a site of PIP2 interaction, we made an in-frame deletion to excise this region (D-HERG) or a mutation to neutralize the charged residues (M-HERG; Figs. 1, 2, and 3). The effects of PIP2 on WT and mutant channels (D- and M-HERG) were compared by functional expression in CHO cells. Intracellular administration of 10 µM PIP2 increased HERG current amplitude (see Fig. 1). Moreover, when the peak amplitudes of the tail currents were normalized, a 19-mV hyperpolarizing shift in voltage-dependent activation was seen (WT Vh: control, 0.99 ± 1.29; PIP2, –18.1 ± 2.3 mV; P < 0.05; n = 8 cells). The PIP2-dependent effects occurred rapidly and lasted at least 12 min. Figure 1D shows the time course of the effect of PIP2 on the tail current amplitude measured at 0 mV. The isochronal I–V relationship during the test pulse also shows a similar hyperpolarizing shift in voltage dependence of both the positive and negative slope conductances (Fig. 1C, inset). The PIP2-induced augmentation of HERG current was most pronounced at voltages negative to 10 mV (Fig. 1C, inset). 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. These data are consistent with our previous findings that PIP2 produced an increase in the voltage sensitivity of HERG K+ channel activation (4).



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  		Fig. 1. Effects of phosphatidylinositol 4,5-bisphosphate (PIP2) on wild-type (WT) human ether-à-go-go-related gene (HERG) K+ channel activation. Currents were obtained immediately after establishment of whole cell configuration (0 min, {circ}) and 3 min after whole cell configuration and internal perfusion of 10 µM PIP2 ({bullet}). A and B: representative current tracings in response to a series of membrane depolarizations in a Chinese hamster ovary (CHO) cell expressing HERG. Currents were elicited by 4.5-s depolarizing steps to various levels (between 50 and –40 mV) followed by repolarizing steps to –40 mV then to –120 mV (B, bottom). C: current-voltage (I–V) relation curve plotted from current measured at the end of depolarizing test pulses (relative current amplitude, I/Imax; during depolarizing steps plotted against the step potential, Imax is the maximal current before PIP2 perfusion). D: voltage-dependent activation curves plotted from peak tail currents during a repolarizing step to –40 mV after depolarization to various voltages. Data were normalized to maximal current (top inset). Time course of the effect of PIP2 on current amplitude at 0 mV (bottom inset) is shown. Values are shown as means ± SE; n = 8 cells; *P < 0.05 vs. data in same voltage at 0 min. E: alignment of WT-HERG shows a clustering of positively charged amino acids (arginines and lysines) in the COOH terminus.

 


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  		Fig. 2. Effects of PIP2 on deletion-HERG (D-HERG) K+ channel activity. Representative current tracings in response to a series of membrane depolarizations in a CHO cell expressing HERG are shown. A: current measured immediately after establishment of whole cell access (0 min) in D-HERG. B: current from the same cells measured 3 min after whole cell access with 10 µM PIP2 in the pipette solution in D-HERG. C: I–V relation curve plotted from current measured at the end of depolarizing test pulses in D-HERG. Data were normalized to unity before and after PIP2 perfusion (inset). D: voltage-dependent activation curves in D-HERG plotted from peak tail currents during a repolarizing step to –40 mV after depolarization to various voltages. Time course of the effect of PIP2 on current amplitude at 0 mV is shown (inset). Values are means ± SE; n = 7 cells. E: partial amino acid sequence of D-HERG shows the segment of positively charged amino acids (arginines and lysines) in the COOH terminus of HERG that were deleted.

 


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  		Fig. 3. Effects of PIP2 on methionine-substituted-HERG (M-HERG) K+ channel activity. Representative current tracings in response to a series of membrane depolarizations in a CHO cell expressing M-HERG. A: current tracings at 0 min. B: current tracings from the same cell 3 min after whole cell access with 10 µM PIP2 in the pipette solution. C: summary data of I–V relationship for cells expressing M-HERG. Data were normalized to unity before and after PIP2 perfusion (inset). D: summary data for voltage dependence of activation curves for cells expressing M-HERG. Data points were plotted from peak tail currents during a repolarizing step to –40 mV after depolarization to various voltages. Values are means ± SE; n = 7 cells. Data were normalized to maximal current (top inset). Time course of the effects of PIP2 on current amplitude at 0 mV are shown (bottom inset). E: partial amino acid sequence of M-HERG shows the segment of positively charged amino acids (arginines and lysines) in the COOH terminus of HERG that were mutated to either neutral or negatively charged amino acids.

 
The electrophysiological effects of PIP2 on HERG channel activity (see Fig. 1) were largely removed in mutant HERG (D- and M-HERG; Figs. 2 and 3). As we have previously shown, increasing [PIP2] by internal perfusion through the patch pipette augments the current amplitude and shifts the voltage dependence of activation leftward (see Fig. 1). Internal perfusion of PIP2 failed to increase the current amplitude in the tail current or shift the Vh value of D-HERG (Vh: control, 12.2 ± 0.11.4 mV; PIP2, 10.3 ± 0.2 mV; n = 7 cells; Fig. 2D) or M-HERG (Vh: control, 4.3 ± 0.3 mV; PIP2, 0.4 ± 0.4 mV; n = 7 cells; Fig. 3D). Upon examination of the isochronal I–V relations of D- and M-HERG, we observed that PIP2 did augment the current amplitude at voltages in the range of the negative slope conductance (Figs. 2C and 3C). This continued augmentation, albeit small compared with WT-HERG, was in the voltage range where inactivation competes with activation to determine the density of outward current (27).

PIP2-dependent regulation of HERG inactivation and activation. We previously showed that the current augmentation in response to PIP2 elevation was in part due to deceleration of inactivation kinetics of HERG channels (4). Voltage-dependent inactivation accounts for the negative slope conductance in the isochronal I–V curve of HERG, which is the only situation where PIP2 appeared to alter D-HERG activity. Accordingly, we investigated whether PIP2 altered the inactivation kinetics of D-HERG. We used voltage-clamp protocols described by Smith et al. (27) to specifically measure voltage dependence of steady-state inactivation. After a prolonged depolarizing pulse to +20 mV to fully activate the channels, we briefly stepped the membrane voltage to various test potentials and then returned to +20 mV (Fig. 4, A–C). During the brief step, the inactivation process rapidly reversed to the steady-state level appropriate to the test potential. The outward tail current observed on returning to +20 mV gives the relative fraction of open channels, which is determined by the degree of steady-state inactivation remaining at the test potential (27). The fraction of channels that deactivate during the brief hyperpolarizing steps was corrected (Fig. 4, A–C, inset) using the procedure described by Smith et al. (27). Our results show that increased [PIP2] significantly enhanced the current amplitude upon release of inactivation in both WT- and D-HERG (Fig. 4, A–C). By normalizing the curves, we see that there is a small but not significant leftward shift. During an inactivation protocol, the time course for the decline in outward current is a measure of the rate of inactivation and is best fit to a single exponential function (Fig. 4, D–F). Increasing the [PIP2] caused a comparable and significant slowing of voltage-dependent inactivation in WT- and D-HERG (Fig. 4, D–F). This suggests that regions of the channel other than the COOH-terminal site (amino acids 883–894) may be affected by changes in PIP2 that result in altered inactivation.



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  		Fig. 4. Effects of PIP2 on the kinetics of inactivation in WT- and D-HERG K+ currents. A: representative HERG current tracing from steady-state pulse protocol. Pulse protocol used for correcting for simultaneous deactivation is shown (inset). Channels were simultaneously activated and inactivated by holding voltage at 20 mV. Brief repolarizing steps to various potentials relieved inactivation before deactivation took place and resulting currents were measured when the voltage was returned to 20 mV (arrow). B and C: summary data of corrected steady-state inactivation graphed as recovered current plotted against the repolarizing step potential showing increased current amplitude and left shift [seen in normalized curves (inset)] in voltage dependence resulting from PIP2 in both WT- and D-HERG (B and C, respectively; n = 7 each). D: representative current tracing from inactivation onset protocol. Activated channels are released from activation as in A, after which the membrane potential is returned to various potentials, and rates of onset of inactivation are taken from the relaxation of the tail currents; {tau}, time constant of onset of inactivation. E and F: summary of voltage dependence of rates of inactivation shows that PIP2 slows the rate of inactivation in a voltage-dependent fashion in either WT-HERG (E) or D-HERG (F). Symbols represent means ± SE; n = 8–13 cells; *P < 0.05.

 
Similar to our previous finding (4), increasing internal [PIP2] by including PIP2 in the whole cell pipette solution resulted in acceleration of HERG activation kinetics (Fig. 5, A and B). For example, at 20 mV, the rate of activation was decreased from 220.3 ± 18.8 to 155.8 ± 20.7 ms by PIP2. Using the same voltage-clamp protocol that examines the rate of activation without concomitant effects of inactivation, we observed no PIP2-dependent acceleration of D-HERG activation (Fig. 5, C and D). Thus voltage dependence of activation and activation kinetics appears to be affected by PIP2 interactions with channel sites between amino acids 883–894, whereas interactions with separate portions of the channel affect inactivation kinetics.



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  		Fig. 5. Effects of PIP2 on the kinetics of activation in WT- and D-HERG K+ currents. Representative current traces from WT-HERG (A) and D-HERG (C) in response to activation protocol. Membrane was held at –80 mV, depolarized to the desired test potential (either 20, 30, 40, or 50 mV) for progressively longer duration, and then repolarized to –120 mV to rapidly remove inactivation. Rate of activation is obtained from the rate of development of tail current. Beginning of establishment of whole cell current (left traces) and 5 min after PIP2 perfusion into the pipette (right traces) are shown. Summary data show PIP2-dependent changes in rate of activation between 20 and 50 mV for WT-HERG (B) and D-HERG (D); n = 9–12 cells.

 
Direct binding of PIP2 with HERG K+ channels. The direct binding of PIP2 with HERG K+ channels was studied using [3H]PIP2. As a positive control for PIP2 binding, we compared results with binding to the inward rectifying channel encoded by Kir2.1, which has been shown to bind to PIP2 (14). Myc-tagged WT-HERG, D-HERG, M-HERG, and Kir2.1 were precipitated using anti-myc antibody. To verify that channel protein was precipitated before we began scintillation counting, 10% of the precipitates were subjected to SDS-PAGE and immunoblot analysis (Fig. 6A, top). As previously shown by Huang et al. (14), Kir2.1 was seen to bind PIP2 by our assay (Fig. 6A, bottom). Specific binding was determined after incubation with 100-fold excess unlabeled PIP2. WT-HERG K+ channel protein also bound [3H]PIP2 and was almost completely competed by excess unlabeled PIP2. D- and M-HERG, however, bound less [3H]PIP2, most of which was nonspecific (Fig. 6A). These data strongly suggest that the polycationic region (amino acids 883–894) is a PIP2 binding site of HERG K+ channels. Dose-dependent competition from increasing amounts of unlabeled data shown in Fig. 6B yielded an apparent Kd of ~0.48 µM.



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  		Fig. 6. Direct binding of PIP2 with HERG K+ channel. A: immunoprecipitated WT-HERG, D-HERG, M-HERG and Kir2.1 channel protein with anti-myc antibody (top). Binding of [3H]PIP2 liposomes (0.3 µM) by WT-HERG, D-HERG, M-HERG, and Kir2.1 (bottom). D- and M-HERG show reduced binding of [3H]PIP2 compared with WT channel protein (open bars). Unlabeled PIP2 (30 µM) reduces the specific binding of [3H]PIP2 to WT-HERG and Kir2.1 but has little effect on D- or M-HERG binding (solid bars), which indicates reduced specific PIP2 binding. Background binding of [3H]PIP2 to beads was subtracted. *P < 0.05 vs. binding of 0.3 µM [3H]PIP2 alone in same group, respectively; #P < 0.05 vs. binding of 0.3 µM [3H]PIP2 alone in WT-HERG group; n = 5–8. B: displacement of the binding of [3H]PIP2 (0.5 µM) to WT-HERG by increasing concentrations of unlabeled PIP2. Each data point represents mean ± SE (n = 4 experiments) of the [3H]PIP2 bound to WT-HERG in the presence of 0, 10–7, 10–6, 10–5, 10–4, or 10–3 M unlabeled PIP2.

 
Effects of {alpha}1A-adrenergic receptor stimulation on IKr in ventricular myocytes. To investigate the IKr properties of rabbit heart, we performed whole cell patch-clamp recordings of isolated rabbit ventricular myocytes. In the whole cell configuration, cells were depolarized from a holding potential of –70 mV to voltages ranging from –40 to 50 mV for 4.5 s. Upon repolarization to –40 mV, an outward current characteristic of IKr was observed (Fig. 7A). Application of the specific IKr blocker E-4031 (1 µM) to the external solution abolished this tail and thereby confirmed its identity as IKr (Fig. 7B). Moreover, 1 µM E-4031 produced a significant prolongation in the action potential duration at 90% repolarization from 501.4 ± 31.2 to 593.0 ± 27.8 ms in cardiac myocytes (Fig. 7C). This is consistent with the previous findings of cardiac IKr (5, 15, 20). To exclude contamination of IKr by coexisting IKs, we also measured IKr in the presence of the IKs inhibitor Chromanol 293B. Figure 7D shows that administration of 10 µM Chromanol 293B had no significant effect on IKr, which indicates the lack of contribution of IKs to the tail currents.



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  		Fig. 7. Pharmacological identification of rectifier K+ current (IKr) in rabbit ventricular myocytes. A: representative current tracings in response to a series of membrane depolarizations in a single isolated rabbit cardiac myocyte. Currents were elicited by 4.5-s depolarizing steps to various levels (between 50 and –40 mV) followed by repolarizing steps to –40 mV. B and D: effects of E-4031 (B) and Chromanol 293B (D) on rabbit ventricular IKr. Voltage-dependent activation curves in IKr plotted from peak tail currents during a repolarizing step to –40 mV after depolarization to various voltages. Control, {circ}; after E-4031, {bullet}; n = 3–5 myocytes. C: E-4031 prolongs rabbit ventricular myocyte action potential duration.

 
IKr amplitude and voltage dependence in rabbit cardiac myocytes were stable for at least 15 min and were not affected by perfusion with bath solution or vehicle (0.5% DMSO; Fig. 8A). There was no significant rundown in the voltage dependence on the activation curve. To test whether physiological receptor-mediated alterations of [PIP2] could affect IKr, the {alpha}1A-adrenergic agonist phenylephrine (PE) was applied ~3 min after the whole cell configuration was established. Application of 10 µM PE resulted in a consistent decrease in the current amplitude (Fig. 8B). The same result was also found in cells pretreated with 10 µM Chromanol 293B (data not shown). To test whether the effect of PE is mediated by {alpha}1A-receptor activation, the selective {alpha}1A-receptor antagonist 5-methylurapidil was used. Pretreatment with 1 µM 5-methylurapidil for 1 h abolished the effect of PE (Fig. 8C), which suggests a receptor-mediated effect. Internal application of PIP2 (10 µM) abolished the inhibitory effect of PE on IKr (Fig. 8D), which suggests that PIP2 may be involved in this effect.



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  		Fig. 8. Activation of a G{alpha}q-coupled {alpha}1A-adrenergic receptor or internal anti-PIP2 monoclonal antibody (PIP2-AB) alters IKr in isolated rabbit cardiac myocytes. Voltage-dependent activation curves before and 30-s after application of 10 µM phenylephrine (PE) were plotted from the tail current amplitude at –40 mV against the depolarizing step voltage (voltage protocol as in Fig. 1A). Representative currents in different treatment groups (left) and group normalized data (right) are shown. A: perfusion with vehicle alone caused no change in activation parameters. B: PE. C: effects of PE in the cells pretreated with the selective {alpha}1A-receptor antagonist 5-methylurapidil (5-MU; 10 µM). D: effects of PE with 10 µM PIP2 in the pipette solution. E: effects of PE in the cells pretreated with the selective PKC inhibitor chelerythrine (1 µM) for 1 h. F: effects of PE with 15 mM BAPTA in the pipette solution in the cells pretreated with chelerythrine. G: effects of internal PIP2-neutralizing antibody (60 nM) on IKr; n = 5–9 myocytes; *P < 0.05 compared with data at same voltages before treatment with PE. Data in voltage activation curves are fitted with a Boltzmann function.

 
We originally reported that voltage dependence of activation was shifted when [PIP2] was altered (4). Despite a decrease in IKr density with PE, there was no shift in Vh (Vh: before PE treatment, 8.59 ± 0.7; after treatment, 8.39 ± 0.67 mV). G protein-coupled receptor (GPCR) stimulation activates PLC and generates DAG from PIP2 hydrolysis, which may activate PKC. Heath and Terrar (12) reported evidence to suggest that activation of PKC could enhance IKr in ventricular myocytes. To examine the possibility that DAG activation of PKC may affect HERG activity during {alpha}-adrenergic stimulation, we pretreated cells with PKC inhibitors. Pretreatment of cells with the PKC inhibitor chelerythrine (1 µM) for 1 h before patch clamping enhanced the effects of {alpha}-adrenergic stimulation and resulted in an even greater PE-induced inhibition of IKr amplitude (Fig. 8E). When PKC was inhibited, subsequent PE treatment produced a reduction in current density and a depolarizing shift in voltage dependence of activation (Vh: before PE treatment, 0.03 ± 0.49; after treatment, 6.51 ± 0.96 mV). Comparable results occurred using another PKC inhibitor, 100 nM BSM (data not shown). These data suggested that PIP2 consumption and PKC activation may alter cardiac IKr in competing directions during {alpha}-adrenergic stimulation and that the PIP2 effect is not due to activation of PKC.

The other second messenger generated by PLC activation is IP3, which upon binding to its receptor releases Ca2+ from the endoplasmic reticulum. Alterations of intracellular Ca2+ may also alter channel behavior. To examine the possibility that {alpha}-adrenergic-mediated regulation of IKr is due to IP3-mediated [Ca2+]i elevation, the Ca2+ chelator BAPTA (15 mM) was included in the whole cell pipette solution to increase the intracellular buffering capacity. In cells pretreated with chelerythrine and BAPTA in the pipette, PE still produced a comparable reduction in current amplitude (Fig. 8F) and a small right shift of the voltage dependence of activation (Vh: before PE treatment, 0.83 ± 1.05; after treatment, 4.47 ± 0.35 mV). These data support the hypothesis that stimulation of cardiac GPCRs that activate PLC can alter endogenous [PIP2] sufficiently to modify the behavior of IKr. Moreover, inclusion of a neutralizing anti-PIP2 monoclonal antibody in the whole cell pipette solution produced a significant reduction of HERG K+ current density (Fig. 8G). When the data were normalized to unity, a 9–10-mV depolarizing shift in voltage-dependent activation was seen (Vh: control, 5.1 ± 1.6; anti-PIP2 monoclonal antibody, 15 ± 2.2 mV; n = 7).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Gq protein-coupled receptor stimulation activates heterotrimeric G proteins with dissociation of G{alpha}q subunits from G{beta}{gamma} subunits. Activated G{alpha}q protein in turn stimulates the activity of phosphatidylinositol-specific PLC. The substrate for PLC is PIP2, a membrane phospholipid that is hydrolyzed to IP3 and DAG. Thus as these GPCRs are activated, there is an increase in the concentrations of second messengers IP3 and DAG with a simultaneous reduction of [PIP2] within the plasma membrane. We previously showed that activation of {alpha}1A-receptors in cells that heterologously express the receptor and HERG with the selective agonist PE altered HERG channel activity in a fashion consistent with reduction of PIP2. These effects were not prevented by internal Ca2+ buffering or PKC inhibition, which is in support of a mechanism whereby GPCR stimulation of PLC results in the consumption of endogenous PIP2 (4). In the present study, we show that stimulation of cardiac {alpha}-adrenergic receptors produced a response in cardiac IKr similar to that seen with heterologously coexpressed HERG and {alpha}1A-adrenergic receptors. One particular difference was noted, however. In cardiac myocytes, the {alpha}-adrenergic-mediated effects on IKr were accentuated by prior block of PKC, which suggests that PKC activation in this situation has an opposing regulation of the channel. The effect of PKC on HERG and IKr, however, remains controversial. Stimulation of the Gq-coupled thyrotropin-releasing hormone receptor, when coexpressed with HERG in Xenopus oocytes, produced kinetic changes that reduced current density; this effect was mimicked by phorbol ester and prevented by the PKC inhibitor FG109203X (2). These findings were also seen in pituitary GH3 cells (11, 24). HERG expressed in Xenopus oocytes was also suppressed by PKC activation via a mechanism that may not involve direct phosphorylation of the channel itself (31). In contrast with these findings, PKC activation in guinea pig cardiac myocytes enhanced IKr through a reduction in C-type inactivation (12). These conflicting effects of PKC on HERG and IKr may result from different signaling systems in different tissues including different differential expression of PKC isoforms or anchoring proteins.

A change in [Ca2+]i may also alter HERG and IKr function. Heath and Terrar (13) found that that the use of Ca2+ buffers reduced {beta}-adrenergic regulation of IKr. In the present study, we found that any changes in [Ca2+]i caused by stimulation of {alpha}-adrenergic receptors were unlikely to play a role in IKr regulation given that 15 mM BAPTA did not alter the PE-induced effects on channel behavior.

HERG is the first voltage-gated channel shown to be affected by changes in [PIP2]; however, the site(s) of interaction with PIP2 has not been determined. More progress has been made in characterizing PIP2 interactions with IKr channels. Clustering of positively charged side-chain amino acids along NH2- or COOH-terminal cytoplasmic channel portions has been proposed as a potential PIP2 interaction site (9). The mechanism of interaction may be an electrostatic attraction between these side chains and the negatively charged polar heads of the phospholipid. We found that the polycationic region from amino acid 883 to 894 is responsible for much of the PIP2 regulation of HERG activity. Despite the elimination of most PIP2 binding by this mutation, there remained some effects on voltage-dependent inactivation. These data suggest that another, possibly lower-affinity binding site for PIP2 may exist within the HERG protein. Multiple PIP2 binding sites in Kir2.1 channels have been proposed in several COOH-terminal loci (25, 28). In the HERG K+ channel, another polycationic segment (672: RYHTQMLRVREFIRFHQIPNPLRQRL697) exists in the cytoplasmic COOH terminus; this may represent an additional site for PIP2 interaction that affects inactivation.

In the IKr channels, the constitutively active Kir2.1 and Kir1.1 interact with PIP2 with high affinity, whereas the G protein-activated Kir3.1/3.4 channels show weaker interactions with PIP2. When exogenous PIP2 is applied, the channels are dynamically activated in the reverse order as follows: Kir3.1/3.4 > Kir2.1 > Kir1.1 (14, 21, 34). It is hypothesized that weaker binding to Kir3.1/3.4 channels allows for appreciable off-rates at physiological concentrations of PIP2, whereas the higher affinity of Kir2.1 (Kd, 0.5 µM; Ref. 14)and Kir1.1 (Kd, 0.58 µM; Ref. 17) allows PIP2 to be constitutively bound (21). Weaker PIP2 binding therefore may produce a situation where physiological consumption by PLC may more readily regulate the channel. Our study showed that the affinity of HERG for PIP2 is comparable to Kir2.1 or Kir1.1, which suggests that the in vivo regulation of HERG by PIP2 may be more similar to these channels. A small reduction or change in the voltage dependence of IKr by changes in PIP2 may have significant impact on repolarization in vivo particularly during periods of extreme stress and autonomic stimuli.

Taken together, our present study demonstrates that alteration of endogenous [PIP2] of cardiac myocytes regulates IKr. The polycationic region (amino acids 883–894) in the COOH terminus of HERG is a physical and functional binding site for PIP2. These findings provide an additional link between cardiovascular stresses, autonomic stimulation, and arrhythmias (both hereditary and acquired). This novel response supports a model where autonomic stimulation of cardiac G protein-coupled receptors may alter IKr-dependent repolarizing forces that may impact propensity toward ventricular arrhythmias.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute (Grant RO1 HS-57388 to T. V. McDonald) and the American Heart Association (AHA) NYC Affiliate (Grant 0120335T to J.-S. Bian and an AHA Established Investigator Award to T. V. McDonald).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. V. McDonald, Depts. of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: mcdonald{at}aecom.yu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Balser JR, Bennett PB, and Roden DM. Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier. J Gen Physiol 96: 835–863, 1990.[Abstract/Free Full Text]
  2. Barros F, Gomez-Varela D, Viloria CG, Palomero T, Giraldez T, and de la Pena P. Modulation of human erg K+ channel gating by activation of a G protein-coupled receptor and protein kinase C. J Physiol 511: 333–346, 1998.[Abstract/Free Full Text]
  3. Baukrowitz T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP, and Fakler B. PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science 282: 1141–1144, 1998.[Abstract/Free Full Text]
  4. Bian J, Cui J, and McDonald TV. HERG K+ channel activity is regulated by changes in phosphatidyl inositol 4,5-bisphosphate. Circ Res 89: 1168–1176, 2001.[Abstract/Free Full Text]
  5. Clay JR, Ogbaghebriel A, Paquette T, Sasyniuk BI, and Shrier A. A quantitative description of the E-4031-sensitive repolarization current in rabbit ventricular myocytes. Biophys J 69: 1830–1837, 1995.[ISI][Medline]
  6. Cui J, Kagan A, Qin D, Mathew J, Melman YF, and McDonald TV. Analysis of the cyclic nucleotide binding domain of the HERG potassium channel and interactions with kcne2. J Biol Chem 276: 17244–17251, 2001.[Abstract/Free Full Text]
  7. Cui J, Melman Y, Palma E, Fishman GI, and McDonald TV. Cyclic AMP regulates the HERG K+ channel by dual pathways. Curr Biol 10: 671–674, 2000.[CrossRef][ISI][Medline]
  8. Curran ME, Splawski I, Timothy KW, Vincent GM, Green ED, and Keating MT. A molecular basis for cardiac arrhythmia: HERG mutations cause long QT syndrome. Cell 80: 795–803, 1995.[CrossRef][ISI][Medline]
  9. Fan Z and Makielski JC. Anionic phospholipids activate ATP-sensitive potassium channels. J Biol Chem 272: 5388–5395, 1997.[Abstract/Free Full Text]
  10. Flanagan LA, Cunningham CC, Chen J, Prestwich GD, Kosik KS, and Janmey PA. The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau. Biophys J 73: 1440–1447, 1997.[Medline]
  11. Gomez-Varela D, Barros F, Viloria CG, Giraldez T, Manso DG, Dupuy SG, Miranda P, and de la Pena P. Relevance of the proximal domain in the amino terminus of HERG channels for regulation by a phospholipase C-coupled hormone receptor. FEBS Lett 535: 125–130, 2003.[CrossRef][Medline]
  12. Heath BM and Terrar DA. Protein kinase C enhances the rapidly activating delayed rectifier potassium current, IKr, through a reduction in C-type inactivation in guinea-pig ventricular myocytes. J Physiol 522: 391–402, 2000.[Abstract/Free Full Text]
  13. Heath BM and Terrar DA. Separation of the components of the delayed rectifier potassium current using selective blockers of IKr and IKs in guinea-pig isolated ventricular myocytes. Exp Physiol 81: 587–603, 1996.[Abstract]
  14. Huang CL, Feng S, and Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. Nature 391: 803–806, 1998.[CrossRef][Medline]
  15. Jurkiewicz NK and Sanguinetti MC. Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent. Circ Res 72: 75–83, 1993.[Abstract/Free Full Text]
  16. Kagan A, Melman YF, Krumerman A, and McDonald TV. 14–3-3 Amplifies and prolongs adrenergic stimulation of HERG K+ channel activity. Embo J 21: 1889–1898, 2002.[CrossRef][ISI][Medline]
  17. Liou HH, Zhou SS, and Huang CL. Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. Proc Natl Acad Sci USA 96: 5820–5825, 1999.[Abstract/Free Full Text]
  18. Loussouarn G, Park KH, Bellocq C, Baro I, Charpentier F, and 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 22: 5412–5421, 2003.[CrossRef][ISI][Medline]
  19. Mitcheson JS, Chen J, and Sanguinetti MC. Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate. J Gen Physiol 115: 229–240, 2000.[Abstract/Free Full Text]
  20. Paquette T, Clay JR, Ogbaghebriel A, and Shrier A. Effects of divalent cations on the E-4031-sensitive repolarization current, IKr, in rabbit ventricular myocytes. Biophys J 74: 1278–1285, 1998.[Medline]
  21. Rohacs T, Chen J, Prestwich GD, and Logothetis DE. Distinct specificities of inwardly rectifying K+ channels for phosphoinositides. J Biol Chem 274: 36065–36072, 1999.[Abstract/Free Full Text]
  22. Sanguinetti MC, Jiang C, Curran ME, and Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81: 299–307, 1995.[CrossRef][ISI][Medline]
  23. Sanguinetti MC and Jurkiewicz NK. Two components of the delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96: 195–215, 1990.[Abstract/Free Full Text]
  24. Schledermann W, Wulfsen I, Schwarz JR, and Bauer CK. Modulation of rat erg1, erg2, erg3 and HERG K+ currents by thyrotropin-releasing hormone in anterior pituitary cells via the native signal cascade. J Physiol 532: 143–163, 2001.[Abstract/Free Full Text]
  25. Shyng SL, Cukras CA, Harwood J, and Nichols CG. Structural determinants of PIP2 regulation of inward rectifier KATP channels. J Gen Physiol 116: 599–608, 2000.[Abstract/Free Full Text]
  26. Shyng SL and Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282: 1138–1141, 1998.[Abstract/Free Full Text]
  27. Smith PL, Baukrowitz T, and Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379: 833–836, 1996.[CrossRef][Medline]
  28. Soom M, Schonherr R, Kubo Y, Kirsch C, Klinger R, and Heinemann SH. Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels. FEBS Lett 490: 49–53, 2001.[CrossRef][ISI][Medline]
  29. Spector PS, Curran ME, Keating MT, and Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides. Circ Res 78: 499–503, 1996.[Abstract/Free Full Text]
  30. Thomas D, Zhang W, Karle CA, Kathofer S, Schols W, Kubler W, and Kiehn J. Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A. J Biol Chem 274: 27457–27462, 1999.[Abstract/Free Full Text]
  31. Thomas D, Zhang W, Wu K, Wimmer AB, Gut B, Wendt-Nordahl G, Kathofer S, Kreye VA, Katus HA, Schoels W, Kiehn J, and Karle CA. Regulation of HERG potassium channel activation by protein kinase C independent of direct phosphorylation of the channel protein. Cardiovasc Res 59: 14–26, 2003.[Abstract/Free Full Text]
  32. Tseng GN. IKr: the hERG channel. J Mol Cell Cardiol 33: 835–849, 2001.[CrossRef][ISI][Medline]
  33. Xie LH, Takano M, Kakei M, Okamura M, and Noma A. Wortmannin, an inhibitor of phosphatidylinositol kinases, blocks the MgATP-dependent recovery of Kir6.2/SUR2A channels. J Physiol 514: 655–665, 1999.[Abstract/Free Full Text]
  34. Zhang H, He C, Yan X, Mirshahi T, and Logothetis DE. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat Cell Biol 1: 183–188, 1999.[CrossRef][ISI][Medline]



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