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1 Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, UK
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Abstract |
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q/11-coupled M3-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nM bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [32P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.
(Received 23 October 2006;
accepted after revision 13 March 2007;
first published online 15 March 2007)
Corresponding author J. S. Mitcheson: University of Leicester, Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University Road, Leicester LE1 9HN, UK. Email: jm109{at}leicester.ac.uk
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Introduction |
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ERG channels are also expressed in neuronal tissue (Papa et al. 2003; Guasti et al. 2005) and may contribute to the maintenance of the resting membrane potential and cellular excitability. Pharmacological inhibition of ERG currents in neuroblastoma cells abolishes spike frequency adaptation during long lasting depolarizations (Chiesa et al. 1997; Selyanko et al. 1999) consistent with slow ERG current activation providing a progressively increasing repolarizing influence. In this regard, ERG currents may limit repetitive firing in a similar manner to M-currents. Indeed, ERG channels are thought to contribute to M-like currents in the brain (Meves et al. 1999; Selyanko et al. 1999) and thus neurotransmitter-mediated modulation of ERG current amplitudes may be important for regulating neuronal excitability. In addition, there is considerable evidence that modulation of ERG channels by thyrotropin-releasing hormone (TRH) results in membrane depolarization that increases the rate of action potential firing and secretion of prolactin (reviewed in Schwarz & Bauer, 2004). Thus ERG channels are expressed in a variety of tissues and receptor-mediated modulation of activity is vital to their physiological function.
There have been several studies on TRH receptor modulation of ERG (Barros et al. 1998; Schwarz & Bauer, 1999; Schledermann et al. 2001; Storey et al. 2002; Bauer et al. 2003; Gomez-Varela et al. 2003b), but relatively little is known about modulation of ERG channels by other G-protein coupled receptors (Selyanko et al. 1999; Kagan et al. 2002; Hirdes et al. 2004; Thomas et al. 2004). Receptor stimulation tends to cause a reduction in maximal current amplitude, a positive shift of activation and acceleration of deactivation, with little or no effect on inactivation. However, there are divergent reports on the underlying signalling mechanisms and the importance of channel phosphorylation. TRH receptor and M1 muscarinic receptor mediated current inhibition has been reported to be largely insensitive to either kinase inhibitors or cell dialysis with non-hydrolysable analogues of ATP (Schledermann et al. 2001; Storey et al. 2002; Hirdes et al. 2004), suggesting phosphorylation is not required. On the other hand, IKr and hERG current modulation by 1A and 
adrenoceptor (AR) stimulation is blocked by inhibitors of protein kinases (Heath & Terrar, 2000; Karle et al. 2002; Thomas et al. 2004). Elevating cAMP to directly activate protein kinase A (PKA) causes a positive shift of activation that is removed when four consensus PKA phosphorylation sites on hERG are mutated (Thomas et al. 1999; Cui et al. 2000). Thus, PKA stimulation alters channel function by a mechanism that requires direct phosphorylation of hERG subunits. The situation with protein kinase C (PKC) dependent modulation is less straightforward. Modulation by phorbol ester activation of PKC remains when 17 of 18 consensus PKC sites on hERG are mutated (Thomas et al. 2003). Although this may indicate that PKC dependent modulation is indirect, perhaps involving PKC phosphorylation of an auxillary channel subunit or signalling molecule (Thomas et al. 2003), mutation of the 18th consensus PKC site (Thr74) produces a non-functional channel – highlighting the importance of this residue and leaving the distinct possibility of direct PKC-mediated phosphorylation at this site.
In the present study, we investigated the modulation of hERG channels by M3-muscarinic receptor stimulation, elevation of the intracellular [Ca2+] ([Ca2+]i), and analogues of diacylglycerol that directly activate PKC. In all cases hERG currents were reduced in a PKC-dependent manner. Direct measurements of subunit phosphorylation indicate that basal phosphorylation is high and is further increased by PKC stimulation. Our results are consistent with receptor-mediated modulation of channel activity by direct PKC phosphorylation of a site on the amino-terminus of hERG.
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Methods |
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HEK-293 cells stably expressing hERG (hERG-HEK cells) were a kind gift from Dr Craig January (University of Wisconsin) and were maintained in Dulbecco's modified Eagle's medium (DMEM) with Glutamax-1, sodium pyruvate, glucose and pyridoxine, supplemented with 10% fetal bovine serum, 400 µg ml–1 geneticin and 50 µg ml–1 gentamycin. Muscarinic receptor modulation of hERG was investigated by transiently expressing wild-type (WT) hERG in HEK-293 cells stably expressing M3 muscarinic receptors (HEK-M3 cells; Tovey & Willars, 2004). hERG mutants (see later) were investigated by transient expression in WT HEK-293 cells. For all transient transfections, 2–5 µg of hERG cDNA was cotransfected with 0.2–0.5 µg EGFP (pEGFP-N1, Clontech laboratories, UK) using Lipofectamine 2000 (Invitrogen, UK), following the manufacturer's instructions. Transfection media were replaced with culture media 6 h after transfection and experiments performed 1–2 days later. Cells were maintained in MEM Alpha Medium without nucleosides (Gibco, UK), supplemented with 10% fetal bovine serum and 100 units ml–1 penicillin and streptomycin (Gibco, UK).
Cloning and mutagenesis of hERG channels
The WT hERG expression construct in pCDNA3.0 and the NTK-hERG expression construct (N-terminus between amino acids 2–354 deleted) in pSP64 were kindly provided by Dr M. Sanguinetti (University of Utah). 4M-hERG, 
PKC-hERG and 18M-hERG constructs in pSP64 were a generous gift from Dr D. Thomas (University of Heidelberg). PKC-hERG lacks 17 of 18 PKC consensus phosphorylation sites, whereas 18M-hERG lacks all 18 (Thomas et al. 2003). 4M-hERG has all four consensus PKA phosphorylation sites mutated. 4M-hERG, PKC-hERG and 18M-hERG were subcloned into pCDNA3.0 for expression in HEK-293 cells. Thr74 mutations were generated from WT hERG using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. All mutations and subcloning reactions were confirmed by DNA sequencing.
Electrophysiology
Whole cell voltage clamp recordings of hERG currents were made using an Axopatch 200B amplifier and Clampex software (Molecular Devices Corp., Sunnyvale, CA, USA). Membrane currents were low pass filtered at 1 kHz and sampled at 2.5 kHz with a Digidata 1320 data acquisition system (Molecular Devices). Borosilicate glass pipettes (Harvard Apparatus, Kent, UK) were pulled and fire polished to get final resistances of 2–5 M
. Series resistances were less than 8 M and were compensated by 60–85%. The pipette solution contained (mM): KCl 130, MgATP 5, Hepes 10, pH 7.2. In some experiments 5 mM BAPTA free acid was added and the pH corrected with KOH. Cells were perfused with extracellular Tyrode solution containing (mM): NaCl 140, MgCl2 1, KCl 4, glucose 10, Hepes 5, CaCl2 2, pH 7.4. A solenoid-based switching system was used to fully exchange solutions in the recording chamber within 60 s and recordings were obtained at 35–37°C. All drug solutions were made up daily to the required concentrations in extracellular solution.
The standard voltage protocol to measure the effects of compounds on hERG current amplitudes was to hold the membrane potential at –80 mV and apply 5 s depolarizations to 0 mV. Tail currents were evoked with 2 or 3 s pulses to –50 mV. The protocol was repeated every 15 s, allowing complete current deactivation between test pulses. The voltage dependence of hERG channel activation was investigated with 5 s depolarizations to test potentials between –40 and +40 mV, applied in 10 mV increments. Peak tail currents upon repolarization to –50 mV were normalized to maximal amplitudes in control solution, plotted against test pulse potential and fitted with a Boltzmann function to obtain half-maximal activation (V0.5,act) values and slope factors. The voltage dependence of steady-state inactivation was measured with a conventional three-pulse protocol. Cells were depolarized to +40 mV for 500 ms, and a 5 ms test pulse to potentials between –140 and +20 mV applied. Current through non-inactivated channels was measured at the beginning of a third pulse to +40 mV, and normalized against the maximal current in each condition. Data were plotted against test pulse potential and fitted with a Boltzmann function to obtain half-maximal inactivation (V0.5,inact) values and slope factors. Electrophysiological recordings were analysed using Clampfit (Molecular Devices) and GraphPad Prism 3.0 (GraphPad Software, SanDiego, CA, USA).
Western blotting of PKC isoforms
hERG-HEK cells were grown to 
70 % confluency in 6-well plates. Down-regulation of PKC isoforms by chronic application of phorbol esters was investigated by incubating cells in either culture medium containing 0.1 % DMSO (control) or medium containing 1 µM phorbol 12-myristate 13-acetate (PMA) for 24 h. In some experiments the inactive 4-PMA analogue was substituted for PMA. Cells were washed once with phosphate-buffered saline (PBS), then lysed for 10 min on ice in solubilization buffer containing (mM) Tris 10, EDTA 10, NaCl 500, 1% Nonidet P-40, 0.5% deoxycholate (pH 7.4), cleared by centrifugation and protein concentrations measured using the Lowry protein assay using bovine serum albumin as a standard (Lowry et al. 1951). Total protein was equal in cells from all wells, including PMA treated cells. For each experimental condition, lysate containing 360 µg total protein was loaded into a full width well of an 8% SDS-PAGE mini gel and proteins separated by electrophoresis, transferred to nitrocellulose and blocked overnight at 4°C in blocking solution containing 5 % (w/v) skimmed powdered milk and 0.1% TWEEN-20 in 0.137 M Tris-buffered saline. Multi-channel blotting apparatus (Mini Protean Multi-Screen, Bio-Rad Laboratories, CA, USA), which divides the nitrocellulose blot into 10 channels (each with equal amounts of protein), was used to simultaneously probe for different PKC isotypes. This system allows detection of multiple PKC isotypes from the same lysate without having to cut the blot into strips. PKC isotype specific antibodies were applied for 1 h at room temperature following manufacturer's instructions (BD Biosciences, NJ, USA; cat. no. 611421). Anti-mouse secondary antibody (Sigma, cat no. A4416), diluted to 1: 1000, was applied for 1 h at room temperature. Protein detection was carried out using ECL + detection (Amersham Biosciences, UK). Control and PMA treated cell samples were prepared in pairs to allow relative levels of each PKC isotype to be compared. To further confirm equivalent protein loading, blots were stripped for 30 min at 50°C in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-Cl, pH 6.7), washed in 0.137 M Tris-buffered saline, blocked for 1 h in blocking solution and re-probed using -actin antibody (Sigma, cat. no. A5441) for 1 h at room temperature.
hERG subunit phosphorylation
Channel phosphorylation in intact cultured cells was carried out using previously described methods (Budd et al. 1999). Briefly, hERG-HEK cells were plated at equal densities onto 6-well plates and grown to 90% confluency. Cells were washed once with phosphate-free Krebs buffer, containing (mM): NaCl 118, KCl 4.3, MgSO4.7H2O 1.17, CaCl2.2H2O 1.3, NaHCO3 0.34, glucose 11.7, Hepes 10 (pH 7.4). Then 5 µCi [32P]orthophosphate (Amersham Biosciences, UK), was added to each well, and the cells incubated for 1 h at 37°C. To stimulate PKC, cells were incubated for 5 min with 10 µM 1-oleoyl 2-acetylglycerol (OAG). In some experiments, PKC was inhibited by incubating cells in bisindolylmaleimide-1 (bis-1) for 15 min prior to OAG stimulation. Bis-1 was at 3 µM used for most experiments, to obtain a rapid and complete PKC inhibition, but we subsequently confirmed in further experiments that 300 nM bis-1 was also sufficient. Alternatively, certain PKC isotypes were down-regulated by culturing cells in medium contained 1 µM PMA for 24 h prior to OAG stimulation. Reactions were terminated by aspiration and addition of 1 ml solubilization buffer. Lysates were cleared by centrifugation and solubilized proteins incubated on ice with 5 µg anti-hERG serum for 90 min. The anti-hERG antibody serum was raised in rabbit against the sequence TCNPLSGAFSGVSNIF (C-terminus hERG residues 983–998) by Pepceuticals, Ltd (Leicester, UK). A previously well characterized pan-hERG1 antibody (Roti et al. 2002), kindly provided by Dr Gail Robertson (University of Wisconsin), was used for immunoprecipitation of transiently expressed WT and NTK-hERG channels. Isolation of immunocomplexes was carried out using protein A-sepharose beads (Amersham Biosciences, UK). Samples were washed with TE buffer containing (mM): Tris-HCl 10, EDTA 10, -glycerol phosphate 20, pH 7.4. Proteins were then resolved on 8% SDS-PAGE gels and stained with Coomassie blue (0.2 % w/v) stain to check for equal antibody loading (and therefore equivalent immunoprecipitation between lanes). Gels were dried and subjected to autoradiography at –80°C for 16–24 h. Phosphorylation was quantified by densitometric measurements of autoradiograph bands using the Alpha Imager 3400 system (Alpha Innotech Corporation, San Leandro, CA, USA). Background was subtracted and values in treated samples were normalized to basal levels in untreated cells from the same experiment.
hERG subunit biotinylation
These experiments were carried out to enable visualization of both the amount of hERG protein and the extent of hERG protein phosphorylation in the same immunoprecipitate. Cells were washed twice with PBS before incubating for 30 min at 37°C in PBS containing 0.5 mg ml–1 sulfo-NHS-LC-biotin (Pierce Biotechnology, Inc., Rockford, IL, USA). Cells were washed and incubated in 10 µCi [32P]orthophosphate per well and the phophorylation assay performed as described above with the exception that lysates were also precleared by incubation on ice with 5 µg rabbit serum (from rabbits in which antibodies have been raised to unrelated protein; mouse muscarinic M3 receptor) and proteins were resolved on a 6% rather than 8% SDS-PAGE gel before transferring to nitrocellulose. The nitrocellulose was blocked overnight, washed and incubated for 30 min at room temperature in 0.5 µg ml–1 streptavidin conjugated with horseradish peroxidase (Pierce) in 0.3 M TBS with 0.1% Tween-20. Excess streptavidin was washed off before protein detection with ECL+. To measure phosphorylation levels of hERG, the nitrocellulose was stripped as described for PKC Western blots, and subjected to autoradiography at –80°C for 48 h.
Phospho-peptide separation by 2D electrophoresis
Isolation of radiolabelled hERG was carried out as described above, with the exception that the amount of [32P]orthophosphate was increased to 200 µCi per well. Following separation on SDS gel, and electroblotting onto nitrocellulose, hERG protein was visualized by autoradiography. The appropriate area of the membrane containing hERG protein was excised and blocked for 30 min at 37°C with 0.5 % polyvinylpyrrolidone-K 30 (Aldrich) solution containing 0.6 % acetic acid, followed by three washes in water and one wash in ammonium bicarbonate solution (50 mM NH4HCO3 and 0.5 mM CaCl2). Control samples from WT HEK cells not expressing hERG were run alongside samples from cells stably expressing hERG and the equivalent area of membrane excised and handled as described above. The samples were then digested with 10 µg ml–1 trypsin in ammonium bicarbonate solution at 30°C for 23 h, and following speed-vac drying and resolubilization in electrophoresis pH 1.9 buffer (formic acid (88 %)/acetic acid/water, 25: 78: 897 v/v), the cleaved peptides were subjected to two dimensional phospho-peptide separation (Boyle et al. 1991) on a cellulose-coated thin layer chromatography plate (Merck, Darmstadt, Germany, 20 x 20 cm). Phospho-peptides were resolved in the first dimension by electrophoresis in pH 1.9 buffer at 2000 V for 30 min using a Hunter HTLE-7002 System (C.B.S. Scientific Company Inc., Del Mar, USA), followed by overnight ascending chromatography in isobutyric acid buffer (isobutyric acid/n-butanol/pyridine/acetic acid/water, 1250: 38: 96: 58: 558 v/v) in the second dimension. The phospho-peptides were visualized with a Storm phosphorimager after an exposure period of 10 or more days.
Measurement of [Ca2+]i
[Ca2+]i was monitored as previously described (Rodrigo et al. 2002). Cells were loaded with 2 µM fura-2-AM (Molecular Probes) for 20 min at room temperature. Cells were alternately excited at 340 and 380 nm with light from a monochromator and emitted light collected at > 520 nm at 2 s intervals from 8 to 16 cells simultaneously using a video imaging system (Photon Technology International, Birmingham, NJ, USA). Data acquistion was performed using PTI Imagemaster software and results expressed as 340/380 ratio of fura-2 emission intensities. Measurements were taken at 35–37°C.
Reagents
Cell culture reagents were purchased from Gibco except for geneticin and gentamycin, which were from Sigma. Ionomycin, methacholine, BAPTA and OAG were all purchased from Sigma. PMA, 4-PMA, bis-1 and Gö6976 were all purchased from Calbiochem.
Statistical analysis
Data values in text and figures represent means ± S.E.M. Error bars are not plotted in figures when smaller than symbols. To test for the significance of reagent effects against untreated cells, Student's t test for paired data was used for biophysical parameters and for unpaired data for other simple comparisons. A one-way ANOVA with post hoc Dunnett's test was used for multiple comparisons. P values less than 0.05 were considered significant.
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Results |
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q/11-coupled M3-muscarinic receptors stably expressed in HEK-293 cells. Activation of M3-muscarinic receptors stimulates phospholipase C, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-phosphate and 1,2-diacylglycerol (DAG). Inositol 1,4,5-phospate rapidly elicits Ca2+ release from the endoplasmic reticulum, whereas DAG initiates longer lasting and slow effects, including the activation of conventional and novel isotypes of PKC. Figure 1 shows the response of hERG currents in HEK-M3 cells to maximal muscarinic receptor stimulation with 1 mM methacholine. hERG currents in the presence of methacholine were smaller at all test potentials (Fig. 1A). The decrease of current amplitude was relatively slow to develop and reached a new steady state level of 73 ± 2 % of control after 4 min (n
= 8, P < 0.005; Fig. 1B). Deactivation of tail currents upon repolarization to –50 mV was significantly faster (P < 0.05, n
= 8); fast and slow time constants (
f and s) were 233 ± 6 ms and 1241 ± 45 ms, respectively, in control solution compared to 170 ± 23 ms and 1021 ± 34 ms in the presence of methacholine. Analysis of voltage-dependent properties revealed a small, but statistically significant (P < 0.05, n
= 5), positive shift in the voltage dependence of activation of 5 ± 1 mV (Fig. 1C), but there was no significant shift in the voltage dependence of inactivation (Fig. 1D). Since hERG is susceptible to block by a wide variety of pharmacological agents, including several G-protein coupled receptor agonists and antagonists, we tested for direct block by methacholine. However, no inhibition of current in hERG-HEK cells (not over-expressing M3-muscarinic receptors) was observed (data not shown), indicating that methacholine acts via a receptor mediated pathway.
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Modulation of hERG by ionomycin and methacholine is dependent on PKC activity
The results described above are consistent with ionomycin activating conventional PKC (cPKC) isotypes and muscarinic receptor stimulation modulating hERG via either conventional or novel PKC isotypes. To investigate whether reduction of PKC activity alters the ability of muscarinic receptor stimulation to regulate hERG we first tested the PKC-selective inhibitor bis-1. High concentrations of bis-1 directly inhibited hERG; 1 µM bis-1 reduced hERG current by > 30%. Similar effects have been reported previously (Thomas et al. 2004). Therefore, we used a concentration of 300 nM bis-1, which did not significantly block hERG channels. Currents were elicited by repetitive pulsing to 0 mV, with peak tail currents recorded at –50 mV; 300 nM bis-1 was applied extracellularly for 3 min and then co-applied with methacholine. In the presence of 300 nM bis-1, the effects of methacholine were substantially attenuated; 85 ± 4% current remained after 3 min of methacholine application, which is not significantly different from current reductions due to rundown in untreated cells over the same time period (Fig. 2). Bis-1 at 300 nM also significantly reduced the response to ionomycin (P < 0.05, n = 5; see Fig. 2B). Therefore, the results with bis-1 suggest a role for PKC in hERG modulation by ionomycin and methacholine.
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, , 
, 
, 
, and 
PKC isotypes (Fig. 3C). In cells pretreated with 1 µM PMA for 24 h, isotypes and were almost undetectable, and protein levels were consistently reduced compared to untreated cells. No changes to isotype expression were observed with 4-PMA, a PMA analogue that does not bind to or stimulate PKC (Fig. 3C). Thus, PKC down-regulation is PMA specific and reduces the levels of Ca2+ and DAG activated isotypes of PKC.
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, or isotypes are implicated, since the hERG current responses were abolished when these PKC isoforms were down-regulated. Modulation of hERG by OAG
A more direct method to investigate modulation of hERG current by PKC without activation of other signalling pathways is to stimulate PKC with the DAG analogue OAG (Fig. 3). hERG currents were elicited with repetitive depolarizations to 0 mV and OAG perfused onto the cells. OAG decreased current amplitudes in a concentration dependent manner (pIC50
= 5.9 ± 0.1, n
4). OAG at 10 µM reduced hERG to 56 ± 3 % (n
= 9) of control after 5 min. Deactivation time constants were significantly accelerated by OAG, from 247 ± 41 ms and 1229 ± 150 ms for f and s, respectively, in control conditions, to 144 ± 15 ms and 782 ± 36 ms, respectively, in the presence of OAG (n
= 6). As with methacholine, there was also a small, but statistically significant shift of the voltage dependence of activation by OAG of 6 ± 2 mV (n
= 6), but steady state inactivation was not significantly shifted (data not shown). The decrease of hERG current amplitudes, shift of activation and acceleration of deactivation by OAG could all be abolished by chronic pretreatment of cells with PMA (but not 4-PMA). The modulation by OAG could also be blocked with 100 nM Gö6976 (Fig. 3C), which is selective for and I PKC isotypes. A 3 min application of Gö6976 alone had no effect, indicating the compound did not directly block the channel pore. However, it significantly reduced the action of OAG (P < 0.001, n
= 5), suggesting that it is and/or I PKC isotypes that modulate hERG.
Does the regulation of hERG by OAG depend upon direct phosphorylation by PKC?
hERG contains 18 putative PKC phosphorylation sites and a number of other serine, threonine and tyrosine residues on intracellular domains that could potentially be phosphorylated to alter channel function. To measure direct phosphorylation of hERG channel subunits, cells were incubated with [32P]orthophosphate for 1 h and the amount of radiolabelled phosphorylated hERG quantified. In lysates from cells expressing hERG, immunoprecipation with an anti-hERG antibody identified two phospho-proteins that were highly phosphorylated under basal conditions and were absent in cells not transfected with hERG (Fig. 4A). These phospho-proteins have the molecular masses predicted for core (135 kDa) and fully glycosylated (155 kDa) hERG subunits and correspond with immature and mature, surface-expressed hERG subunits, respectively (Zhou et al. 1998). Tryptic digests of [32P]orthophosphate labelled hERG and the separation of phospho-peptides following 2D electrophoresis suggest that hERG is phosphorylated on a large number of sites (Fig. 4B). Each ‘hot spot’ represents phosphorylated fragments of hERG since identically prepared samples from WT HEK cells produced no detectable phospho-proteins.
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The OAG dependent increase of phosphorylation could be abolished by incubating cells with bis-1 for 15 min or by chronic pretreatment of cells with PMA (Fig. 4E, F and G). Interestingly, bis-1 and chronic pretreatment with PMA had no effect on phosphorylation levels in untreated cells. One conclusion from this may be that PKC does not have a role in basal hERG phosphorylation; however, we cannot entirely discount the possibility that changes in basal phosphorylation at PKC sites are not detectable because of a high level of background phosphorylation by other kinases. These results demonstrate that hERG is constitutively phosphorylated within intact cells and that OAG increases direct phosphorylation of the channel in a PKC dependent manner.
The N-terminus of hERG is required for hERG current modulation and phosphorylation by PKC
Previous mutagenesis studies have shown that PKC-dependent modulation of hERG currents in Xenopus oocytes is not ablated in PKC-hERG, a channel in which 17 of the 18 putative phosphorylation sites have been mutated to Ala (Thomas et al. 2003). OAG also inhibited PKC-hERG and 4M-hERG (PKA phosphorylation sites mutated) currents expressed in HEK-293 cells (Fig. 5A). The percentage inhibition in both mutants was similar to WT hERG. Thus, mutation of all the PKA phosphorylation sites and 17 of 18 PKC sites failed to prevent the channel modulation by OAG.
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PKC-hERG or 18M-hERG (all 18 PKC sites mutated to Ala). Four different anti-hERG antibodies, including the anti-hERG serum and pan-hERG1 antibody, were unable to immunoprecipitate phospho-proteins or detect bands of the anticipated molecular mass in Western blots. Evidently, the mutations prevent the antibodies recognizing their epitopes. As an alternative to mutating Thr74 and other putative phosphorylation sites, we investigated PKC-dependent modulation and phosphorylation of NTK-hERG channels in which amino acids 2–354 (including the PAS) domain have been removed (Spector et al. 1996). NTK-hERG transiently expressed in HEK-293 cells gave functional currents with rapid deactivation kinetics (Fig. 5B). However, whereas WT channel currents recorded on the same experimental day were inhibited by 37 ± 3% (n = 5), a 5 min application of 10 µM OAG failed to significantly inhibit NTK-hERG currents (Fig. 5A and B). The loss of functional effects on NTK-hERG currents correlated with an inability of OAG to alter the phosphorylation state (Fig. 5C). OAG at 10 µM failed to increase NTK-hERG phosphorylation above basal levels, although WT hERG subunit phosphorylation, measured at the same time, increased by 15 ± 2% (n = 5). These results indicate that an intact N-terminus is required for PKC-dependent phosphorylation and modulation of hERG currents, suggesting that critical residues for PKC phosphorylation reside on the N-terminus. The observation that hERG channels that lack an intact N-terminus are not phosphorylated or functionally modulated by PKC activation provides further evidence for the link between direct PKC-dependent phosphorylation of hERG subunits and the regulation of channel activity.
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Discussion |
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, and to a reduced extent isotypes of PKC. The effects of ionomycin and muscarinic receptor stimulation are also closely mimicked by the DAG analogue, OAG, and can be attenuated by bis-1 and preincubation with PMA. Therefore, our findings are entirely consistent with muscarinic-receptor stimulation being coupled to activation of DAG-sensitive isotypes of PKC.
Buffering intracellular Ca2+ with BAPTA does not attenuate the hERG current response to M3-muscarinic receptor stimulation suggesting that a rise of Ca2+ does not directly regulate the channel and also is not obligatory for the PKC mediated modulation. Despite this, the ionomycin response is abolished by BAPTA. The most likely explanation is that cPKC isotypes (activated by DAG in methacholine-treated cells and Ca2+ in ionomycin treated cells) are responsible for the hERG channel modulation. This is further supported by experiments with the and 1 PKC isotype-selective inhibitor Gö6976, which abolished the inhibition of hERG currents by OAG stimulation of PKC. Nevertheless, a role for novel isotypes such as PKC cannot be discounted.
Previous studies on the M1-muscarinic receptor modulation of ERG channels expressed in mammalian cells have also shown the same principal effect – a partial reduction in available current with minimal effects on the voltage dependence of activation and inactivation (Selyanko et al. 1999; Hirdes et al. 2004). M1 and M3 muscarinic receptors are coupled to phospholipase C by the Gq/11 family of G proteins. A recent, detailed study by Hirdes et al. (2004) concluded that although rat ERG1 current modulation by muscarinic receptor stimulation was mediated by phospholipase C, its classical downstream messengers, Ca2+ and particularly PKC, were not involved. Thus, 1 µM bis-1 or staurosporine was ineffective at preventing muscarinic receptor-mediated current inhibition. Despite this evidence suggesting a lack of effect of PKC, the PKC inhibitor calphostin C partially reduced the effects of muscarinic receptor stimulation. Furthermore, the current rundown in the experiments with bis-1 and staurosporine was particularly fast (see Hirdes et al. 2004, Fig. 7), making it difficult to precisely quantify the extent of inhibition. The concentrations of bis-1 and staurosporine used in these experiments were sufficient to block ERG channel conduction and it is possible that the decrease of current described as rundown might actually be use dependent accumulation of block. In the study of Hirdes et al. (2004), dialysis of cells via the patch pipette with a non-hydrolysable analogue of ATP to prevent kinase-mediated phosphorylation events was also ineffective at preventing muscarinic receptor-mediated regulation of ERG1. However, in such protocols it is difficult to verify that levels of ATP are sufficiently depleted within the local vicinity of channels. Interestingly, Cayabyab et al. (2002) did observe more rapid rundown of ERG currents in cells dialysed with 0 mM than 2 mM ATP, suggesting that ATP was required to maintain phosphorylation and normal channel function. The apparent disparity in receptor–channel coupling mechanisms observed in the two studies may also be associated with specific differences in muscarinic receptors (M1
versus M3), channel (rat compared to human ERG) or recording temperatures (35–37°C in this study). In the present study, evidence for the role of PKC comes not only from pharmacological manipulation of PKC activity, but also from direct measurements of changes to phosphorylation.
PKC mediated modulation of hERG
Whether hERG is directly modulated by PKC or not has been controversial. Most studies have activated PKC by acute application of PMA with variable results. In one study in tsA-201 cells, 500 nM PMA had little effect (Hirdes et al. 2004). In other studies, PMA caused a positive shift in the voltage dependence of activation and an acceleration of deactivation, with little reduction in maximal current (Barros et al. 1998; Kiehn et al. 1998; Schledermann et al. 2001). Interestingly, the response to PMA is not abolished by mutating 17 of the 18 consensus PKC phosphorylation sites (see later) and is relatively insensitive to PKC inhibitors (Schledermann et al. 2001). Thus 1 µM bis-1 has little effect (Kiehn et al. 1998; Schledermann et al. 2001) and 10 µM bis-1 is required, with long incubation times, to significantly reduce the response to PMA (Barros et al. 1998; Thomas et al. 2003). At these high concentrations there is direct block of the channel pore and inhibition may not be kinase specific (Davies et al. 2000). Indeed, PKA antagonists and broad spectrum protein kinase inhibitors are also able to block the actions of PMA in some Xenopus oocyte studies (Kiehn et al. 1998; Thomas et al. 2003). This suggests that in addition to PKC, PKA may also be part of the signalling cascade that leads to PMA-mediated hERG current modulation. PMA may also exert its action through other molecules with a DAG binding site.
In the present study we used the DAG analogue OAG to activate PKC. We observed a decrease of maximum available current with little change in voltage dependence of activation or deactivation kinetics. The response to OAG was identical in mutant (4M-hERG) channels without PKA phosphorylation sites, ruling out a role for PKA. The response to OAG mimicked ionomycin and muscarinic receptor stimulation, could be blocked by a relatively low concentration of bis-1 (300 nM), and was also abolished in cells chronically pretreated with PMA. These results strongly suggest a PKC-mediated modulation of hERG current without any contribution from PKA.
Is PKC-dependent modulation due to direct phosphorylation of hERG?
Previous studies have suggested that PKC-mediated modulation of hERG occurs through an indirect mechanism involving an intermediate protein or auxillary channel subunit that is phosphorylated and consequently alters the gating behaviour of the channel. This model was proposed on the basis that the hERG current response to PMA and 1-adrenoceptor stimulation is retained in the PKC-hERG mutant in which 17 of 18 consensus PKC phosphorylation sites have been removed (Thomas et al. 2003, 2004). Nevertheless, a limitation of the mutagenesis approach is that PKC phosphorylation could still occur at the 18th consensus PKC site (Thr74), which cannot be mutated without losing functional expression. In addition, PKC phosphorylation at atypical phosphorylation sites cannot be ruled out. In this study we obtained direct measurements of phosphorylation by [32P]orthophosphate labelling of hERG in intact cells. OAG increased hERG subunit phosphorylation in a PKC-dependent manner. The increase of phosphorylation above basal levels was abolished by incubating cells with bis-1 and chronic pretreatment with PMA. Thus, our results strongly suggest that hERG subunits are directly phosphorylated in response to PKC activation. Deleting the N-terminus of hERG prevents the OAG-mediated modulation of hERG and the increase of phosphorylation, consistent with a role for Thr74. Unfortunately, mutation of Thr74 to Val, Asp or Glu also caused a loss of hERG channel function, indicating that this position on a surface loop of the PAS domain is important for channel function.
A possibility that we cannot exclude is that the kinase that phosphorylates hERG is not PKC, but an intermediary kinase that requires PKC activation. Regardless of the precise position of the phosphorylation site(s) and kinase, our results indicate that hERG channel subunits are directly phosphorylated by a PKC-dependent mechanism and an intact N-terminus is required for modulation of channel function (Gomez-Varela et al. 2003a). The N-terminus could be required for PKC binding or be necessary for allosteric regulation of phosphorylation.
Phosphorylation is an important mechanism for regulating hERG channel activity
Our studies reveal that hERG channel subunits in intact cells are highly phosphorylated under basal conditions. Phospho-proteins with molecular masses corresponding to both core glycosylated and fully glycosylated channels are immunoprecipitated indicating that surface and intracellularly located hERG channels are phosphorylated. Thus, phosphorylation is not restricted to surface localized channels alone. More than 17 phospho-proteins can be isolated in hERG protein that has been purified and digested with trypsin (phosopho-peptide separation), indicating that hERG is phosphorylated at multiple sites. The change of phosphorylation we observed with OAG stimulation was modest. This may arise because only a fraction of channels are phosphorylated by PKC. A more likely explanation, supported by the phospho-peptide analysis, is that the relatively small increase of total phoshorylation by PKC is masked by the high basal level of phosphorylation. Thus, a much larger proportion of the channels may well be phosphorylated by PKC than is indicated by the percentage change in total phosphorylation.
Recently two kinases, protein kinase B (Zhang et al. 2003) and Src tyrosine kinase (Cayabyab & Schlichter, 2002), have been reported to tonically modulate hERG in a manner that would be consistent with basal phosphorylation of the channel. It is becoming apparent that endogenously expressed ERG can exist in a signalling complex in which channel function can be regulated by both tyrosine kinases and phosphatases (Cayabyab & Schlichter, 2002; Cayabyab et al. 2002). Two observations suggest that PKC does not have a profound effect on basal phosphorylation of hERG. First, acute application of 300 nM bis-1 to patch-clamped cells does not significantly alter hERG current amplitudes. Second, the amount of basal phosphorylation is not reduced by 3 µM bis-1 or chronic PMA pretreatment of cells. Thus, significant changes of basal phosphorylation are not obtained with either short or long-term decreases to PKC activity.
This study provides strong evidence that hERG is phosphorylated by PKC (or a kinase dependent on PKC activity), and this leads to inhibition of channel function. Mutagenesis studies indicate that the same applies for PKA, since mutation of four consensus PKA sites prevents the rightward shift of activation and decrease of current density in response to PKA stimulation (Thomas et al. 1999; Cui et al. 2000). Phosphorylation by PKA and PKC provides the potential for hERG to be modulated by a variety of G-protein coupled receptors, and this has important implications for regulating the activity of excitable cells and needs to be further investigated in vivo in cardiac myocytes and neurons. It is becoming apparent that there are many signal transduction pathways that can modify hERG channel function. Our study also demonstrates constitutive phosphorylation at multiple sites under basal conditions. A challenge for the future is to identify the sites of phosphorylation and determine their functional importance in tissues in which hERG is endogenously expressed.
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significantly different from Mch-only response (P < 0.01); 
significantly different from IM-only response (P < 0.05). Note, for clarity not all significant differences are presented.
