Am J Physiol Cell Physiol Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 289: C543-C556, 2005. First published April 27, 2005; doi:10.1152/ajpcell.00025.2005
0363-6143/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/3/C543    most recent
00025.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, S. G.
Right arrow Articles by Leaney, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, S. G.
Right arrow Articles by Leaney, J. L.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

PKC-{delta} sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells

Sean G. Brown, Alison Thomas, Lodewijk V. Dekker, Andrew Tinker, and Joanne L. Leaney

Department of Medicine, University College of London, London, United Kingdom

Submitted 20 January 2005 ; accepted in final form 12 April 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-gated inward rectifier (Kir3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-{delta} mediates channel inhibition because recombinant PKC-{delta} inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-{delta} constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-{delta} to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-{delta} after M3 receptor activation in HEK-293 cells.

phosphatidylinositol 4,5-bisphosphate; phorbol 12-myristate 13-acetate; receptor for activated C kinase; A kinase anchoring protein; carbachol; 5'-N-ethylcarboxyamidoadenosine

INWARDLY RECTIFYING K+ channels, which are regulated by G proteins (the GIRK family of channels) have important physiological roles in the heart and in the brain (44). In atrial myocytes, their activation underlies vagal-mediated bradycardia while in the central nervous system, and in some neuroendocrine cells, they are important in maintaining the resting membrane potential and inhibiting hormone release, respectively (44). Since the molecular identification of the cloned counterparts of these channels (the Kir3.1–3.4 family), many investigators have focused on the molecular mechanisms that underlie the activation of this family of channels, i.e., direct binding of G{beta}{gamma}-subunits in response to stimulation of Gi/o-coupled receptors, such as the Adenosine A1 receptor (25, 26, 38, 49). In more recent years, we and others have attempted to elucidate the mechanisms of channel inhibition after stimulation of Gq/11-coupled receptors. Stimulation of these receptors and thus activation of phospholipase C results in 1) hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), and 2) activation of protein kinase C (PKC). Both PIP2 and PKC have been proposed as important regulators of channel activity (4, 5, 17–19, 27, 28, 30, 33, 34, 42, 47, 52). A question that remains is to the relationship between PIP2 and PKC-mediated channel regulation.

In our previous work (27), we have proposed that a Ca2+-independent PKC isotype inhibits Kir3.1/3.2A channels after stimulation of Gq/11-coupled, muscarinic M3 receptors, and that subsequent activation of the channels by co-expressed Gi/o-coupled adenosine A1 receptors is prevented. In this report, we have used a combination of electrophysiological, biochemical, and imaging techniques to investigate this phenomenon further. We show that purified PKC directly inhibits channel activity when applied to excised patches and we elucidate the specific PKC isotype responsible. Furthermore, we find that PIP2 also has a prominent role in mediating channel inhibition, which is dependent on PKC activity. Many have argued that either PKC or PIP2 regulate the channel: in this study, we provide evidence for a dual regulation of the channel by both PIP2 and PKC. Crucially, we hypothesize and provide evidence for a coordinated mechanism of channel inhibition, centered on the action of PKC, after Gq/11-coupled receptor activation. Furthermore, we identify the isoform of PKC involved in channel inhibition as PKC-{delta}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and molecular biology. Human embryonic kidney (HEK)-293 cell culture and transfection procedures were as previously described (25, 26). We used stable cell lines expressing the Kir3.1/3.2A channel complex alone (HKIR3.1/3.2A) and with either the A1 receptor (denoted as HKIR3.1/3.2A/A1) or the M3 receptor (HKIR3.1/3.2A/M3). We generated a stable cell line expressing Kir3.1/3.2C by subcloning the cDNA for these subunits into the dual promoter vector pBudCE4.1 (Invitrogen) and, after transfection, growing cells under selective pressure (364 µg/ml Zeocin). Mutagenesis of cDNA was done using the QuikChange site directed mutagenesis kit (Stratagene). The myc-tagged PKC isotypes were generated as follows: for mycPKC-{alpha}, an NH2 terminal NcoI fragment of PKC-{alpha} (36) was cloned in frame with the myc tag into pEF-link-TAG. The PKC-{alpha} COOH terminus was spliced into this construct using the unique BamHI site to generate the full-length tagged molecule. A similar strategy was employed for mycPKC-{beta}I, where the NH2 terminal NcoI fragment was taken from pACY1-PKC-{beta}I (7) and the COOH terminus spliced into this construct using the unique internal NotI site. For mycPKC-{gamma} a full-length NcoI-XbaI PKC-{gamma} fragment (6) was cloned directly into pEF-LINK-TAG. For mycPKC-{delta} an NH2 terminal BamHI-BstNI fragment from pKSII+-{delta}(1–298) (8) was cloned in frame with the myc tag into pEF-link-tag. A COOH terminal fragment was spliced into the resulting construct using the unique internal BstE11 site to generate full-length mycPKC-{delta}. For mycPKC-{epsilon}, a 900-bp NcoI-BamHI PKC-{epsilon} fragment (39) was cloned into pEF-link-TAG. The COOH terminus of PKC-{epsilon} was spliced into this construct using the BamHI site. For mycPKC-{eta}, a pBluescript construct was generated with a modified start codon to contain an NcoI site (9). An NH2 terminal NcoI fragment was taken from this construct and cloned into pEF-link-TAG. The COOH terminus of PKC-{eta} was spliced in using the unique internal NheI site. The mycPKC-{zeta} construct has already been described (23). Expression of all cDNA constructs in this study was achieved through transfection with the use of Lipofectamine reagent (Invitrogen), as previously described (25, 26).

Electrophysiological recordings. Whole cell currents were recorded as previously described (25) and we also performed cell-attached and inside-out patch recordings. Currents were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz, digitized at 5 kHz, and data acquired with the use of a Digidata 1322A and pCLAMP8 software (Axon Instruments). Single-channel data was analyzed using Fetchan 6.0 software (Axon Instruments) to generate events lists. The mean number of open probability channel (NPo) values was determined from 30-s sweeps at the appropriate test potential in each experimental condition.

For whole cell recordings, the bath solution contained (in mM) 140 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4) and the pipette solution contained (in mM) 107 KCl, 1.2 MgCl2, 1 CaCl2, 10 EGTA, 5 HEPES, 2 MgATP, and 0.3 Na2GTP (KOH to pH 7.2, ~140 mM total K+). The pipette solution used in inside-out single channel experiments was identical to the whole cell bath solution as described above, whereas the inside-out single-channel bath solution contained (in mM) 140 KCl, 2.6 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.2). For cell-attached experiments, both pipette and bath solutions were identical to whole cell bath solution. For perforated-patch recordings the pipette solution contained (in mM) 140 KCl, 2.6 CaCl2, 1.2 MgCl2, 5 HEPES (pH 7.4), and was supplemented with 240 µg/ml amphotericin B. Drugs were bath applied using either a gravity-fed system or a localized application system (model MSC160; Bio-Logic). Data are presented as means ± SE (unless otherwise stated), where n indicates the number of cells/patches recorded from. Data were analyzed for statistical significance using one-way repeated-measures ANOVA tests with post Bonferroni or Dunnett's test as appropriate (P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001).

Purification of PKC. PKC was purified by following a method modified from Allen et al. (1). Briefly, 250–300 g wt adult Sprague-Dawley rats were euthanized by cervical dislocation in accordance with Home Office Guidelines for Animals (Scientific Procedures) Act 1986. The brains were removed and the hippocampi extricated into ice-cold PBS. The brain tissue was stored in cryovials at –80°C and used within 2 wk. All subsequent procedures were carried out at 4°C. Frozen tissue was thawed on ice and homogenized using a glass-on-glass Dounce homogenizer in solution A, which contained 20 mM Tris·HCl, 10 mM dithiothreitol (DTT), 1 mM CaCl2, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). This was then centrifuged at 35,000 g for 20 min and the supernatant separated and discarded. The pellet was then resuspended in solution B (identical to solution A, except that CaCl2 was reduced to 0.1 mM) and centrifuged again at 35,000 g for 20 min. This procedure was repeated twice. The pellet was resuspended and homogenized in solution C composed of 20 mM Tris·HCl, 10 mM DTT, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM PMSF, stirred at 4°C for 1 h, and then centrifuged at 100,000 g for 60 min. The resultant supernatant was then applied to a DEAE-Sephacel column preequilibrated with 20 mM Tris·HCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA, and 10% glycerol (solution D). The column was washed with solution D and proteins bound to the column eluted with a linear 0–0.4 M NaCl gradient collecting 5-ml fractions at a flow rate of 10 ml/h. Fractions containing kinase activity were adjusted to 2.25 mM NaCl, 2 mM DTT, 50 µg/ml leupeptin, 10 µg/ml pepstatin A, and 0.5 mM PMSF, and applied to a second column containing preequilibrated phenyl-Sepharose. Bound proteins were eluted using a linear 1.5–0 M NaCl gradient at a flow rate of 5 ml/h, and 2-ml fractions were collected. Fractions were assayed for PKC activity using the PepTag kit (Promega), which allows the detection of purified and active protein kinases on the basis of separation of phosphorylated and nonphosphorylated species on an agarose gel.

Purification of G protein {beta}{gamma}-subunits. A His6 epitope was inserted at the NH2 terminus of G{beta}1 to yield G{beta}1-His6. This and G{gamma}2 were cloned into the pBUDCE4.1 vector and used to create stable HEK-293 cell lines. HEK-293 cells, containing G{beta}1-His6{gamma}2 were harvested in 1x PBS, and the resulting pellet resuspended in lysis buffer A (10 mM HEPES, pH 7.4, 0.5 mM EDTA, and complete protease inhibitors) and left for 20 min on ice. One milliliter of resuspension buffer B (10 mM HEPES pH 7.4, 500 mM sucrose, 0.5 mM EDTA, and complete protease inhibitors) was added and mixed before the cells were fragmented with the use of a Dounce homogenizer. The debris was pelleted at 1,000 g for 15 min. The membranes were pelleted by ultracentrifugation (100,000 g for 60 min), followed by resuspension in buffer C (20 mM HEPES, pH 8.0, 100 mM KCl, complete protease inhibitors, and 1% CHAPS). The homogenized solution was left for 1 h on ice, followed by ultracentrifugation (100,000 g for 1 h). The resulting supernatant was mixed with preequilibrated Ni-NTA resin (Qiagen) on a rotary shaker for 2 h at 200 rpm. The material was loaded into a column and washed with buffer C + 10 mM imidazole for 8-column volumes. G{beta}1-His6{gamma}2 was eluted with 100 mM imidazole (4 column volumes) and the resulting fractions pooled. Imidazole was removed by dialysis and the protein identified by Western blot analysis using the anti-G{beta} antibody (Chemicon) or the anti-pentaHis antibody (Qiagen).

PKC translocation. For membrane localization experiments cells were washed in PBS and Dounce homogenized in ice-cold extraction buffer (20 mM Tris-Cl, pH 7.4, 2 µg/ml aprotinin, 100 µM tosyl-lysine-chloromethyl ketone, 1 µM pepstatin, 50 µg/ml PMSF, and 1 µg/ml diisopropylfluorophosphate). The extract was incubated at 4°C for 15 min and then centrifuged (14,000 rpm, 4°C). The supernatant was taken ("cytosol") and the pellet was reextracted as above. After clearance, the supernatant was discarded and the pellet was homogenized in extraction buffer containing 1% Triton X-100. The homogenate was incubated at 4°C for 30 min and centrifuged as above. The supernatant was taken ("membrane"). One-fifth volume of 5x Laemmli buffer was added to cytosol and membrane fractions and 10 µl of this buffer were analyzed by SDS-PAGE and Western blot analysis.

Western blot analysis. For Western blot analysis, proteins were analyzed by 9% SDS-PAGE and transferred to nitrocellulose as described (48). Nitrocellulose filters were probed and developed using the ECL Western detection system (Amersham) and quantified using ScionImage software (Scion).

Confocal fluorescence microscopy. We used confocal microscopy to examine the translocation of green fluorescent protein (GFP)-tagged PKC-{delta} and PKC-{epsilon} (30) in HEK-293 cells. Cells were imaged 24-h post-transfection at 37°C on a heated stage (Peltier MicroIncubator System) using a Radiance 2000 laser scanning confocal microscope (Bio-Rad). Excitation was measured with the use of a 488-nm laserline and emission was measured at >530 nm. Drugs were bath applied with the use of a gravity-driven perfusion system via a heated perfusion tube connected to a temperature controller (Scientifica). Images were acquired and analyzed using Bio-Rad software (Lasersharp and Laserpix, respectively).

Expression purification and phosphorylation of maltose-binding protein-fusion proteins. The cytoplasmic COOH termini of Kir3.1 (amino acids 180 to 501) and Kir3.2A (amino acids 194 to 425) were cloned into pMALc2 x (New England Biolabs) to create NH2 terminal fusion proteins with the maltose binding protein (MBP). Expression of the fusion proteins in Escherichia coli BL21(DE3) cells was induced using 0.3 mM isopropylthiogalactoside and incubated overnight at 25°C. Cells were harvested by centrifugation at 4,000 g for 30 min at 4°C. The resulting pellet was resuspended in column buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA) containing protease inhibitors (Roche protease inhibitor cocktail, EDTA free) and stored at –20°C until required. Cells were thawed and sonication (5 x 60 s) was used to lyse the cell membranes. After centrifugation (10,000 g for 30 min at 4°C), the supernatant was bound to amylose resin (New England Biolabs) preequilibrated with column buffer. The column was washed with 10 column volumes of column buffer before the protein was eluted with column buffer containing 10 mM maltose. Protein containing fractions were pooled and dialyzed against storage buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM DTT, and 1 mM EDTA). Protein concentration and purity were determined using the Bradford assay and SDS-PAGE, respectively. The phosphorylation assay was carried out as described previously (37).

Materials. Cell culture materials were from Invitrogen. Molecular biology reagents were obtained from New England Biolabs or Roche Molecular Biochemicals. All chemicals were from Sigma, Tocris, or Calbiochem. Drugs were made up as concentrated stock solutions in ethanol, water, or DMSO, and kept at 4°C or –20°C.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We (27) have previously reported that stimulation of the Gq/11-coupled M3 muscarinic receptor led to a profound and long-lasting inhibition of whole cell Kir3.1/3.2A currents and proposed that this was due, in part, to the activation of PKC. In the present study, we observed that M3-mediated channel inhibition exhibited some differences in the dynamics of channel inhibition in perforated-patch mode. In both whole cell and perforated-patch modes, M3 receptor stimulation by carbachol (10 µM) resulted in similar magnitudes of channel inhibition (whole cell: 78.7 ± 2.7%; perforated patch: 66.5 ± 4.2%, P > 0.05, n = 12; Fig. 1A) but that observed in perforated patch was reversible after 5 min, whereas in whole cell, channels remained inhibited for ~20 min (27). Because we have proposed an involvement of PKC in this inhibitory phenomenon, we pretreated cells with the PKC inhibitor staurosporine (1 µM for 5 min) before being used in perforated-patch experiments. Under these conditions, stimulation of M3 receptors no longer resulted in channel inhibition (5.6 ± 1.9% decrease at steady-state current postcarbachol wash, n = 10; Fig. 1B), confirming the involvement of PKC in channel inhibition.



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 1. Inward rectifying K+ channel (Kir3.1/3.2A) currents undergo a reversible form of inhibition under perforated patch configuration, which is dependent upon the activity of protein kinase C (PKC) and phosphatidylinositol kinases. A,i: representative example of the reversible inhibitory effect of M3 receptor stimulation on Kir3.1/3.2A channels. Note that the currents are initially potentiated then significantly inhibited after removal of carbachol (10 µM, CCh) but that channel activity recovers to a level not significantly different from control within 5 min. The point of minimum channel activity after receptor stimulation is marked as "overshoot." Control channel activity is measured at the start of the trace at t = 0. The dashed line indicates zero current. ii: bar chart summarizing the effect of M3 receptor stimulation on Kir3.1/3.2A channel activity studied under the perforated patch configuration (n = 12). *P ≤ 0.05. B: cells preincubated with staurosporine (100 nM) are not inhibited by activation of the M3 receptor in the perforated patch configuration. A typical example is shown in i and data are summarized in ii, n = 10, **P ≤ 0.01. C: Kir3.1/3.2A currents become irreversibly inhibited after M3 receptor stimulation in cells preincubated with wortmannin (10 µM) studied under the perforated patch configuration. A typical example is shown in i and data are summarized in ii, n = 11; ***P ≤ 0.001.

 
We investigated the mechanism of recovery from channel inhibition observed in perforated patch by preincubating cells with wortmannin (10 µM, 10 min), which has been used to demonstrate the involvement of phospholipids in the regulation of other channels (46). After stimulation of the M3 receptors, the channels were still inhibited (66.4 ± 7.3% inhibition; Fig. 1C), and, similarly to that observed under the whole cell, they remained so for the period of recording (8 min). This would suggest that PIP2 is involved in the recovery from inhibition.

We wanted to examine whether channel activity can be restored or rescued by PIP2. We made whole cell recordings from HKIR3.1/3.2/M3 cells using either control or PIP2-containing pipette solution. Under control conditions, in the absence of added PIP2, the magnitude of M3-mediated inhibition was 77.9 ± 2.2% (n = 36). However, in the presence of PIP2, this effect was significantly reduced by about one-half (M3-mediated inhibition: 39.6 ± 7.2%, n = 12, P = 0.0002), suggesting that PIP2 does play a part in the regulation process. To further investigate this rescuing effect of PIP2 we also performed a series of experiments using the HKIR3.1/3.2/M3 cell line co-expressing Gi/o-coupled A1-adenosine receptors. We have previously reported that stimulation of the M3 receptor prevented subsequent channel activation by the A1 receptor (27). In the current study, with the inclusion of PIP2 in the pipette the A1 response was rescued (Fig. 2).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 2. Phosphatidylinositol 4,5-bisphosphate (PIP2)-mediated rescue of Gi/o signaling with Kir3.1/3.2A channels after M3-receptor stimulation. A: M3 receptor was transiently transfected into the HKIR3.1/3.2A/A1 cell line and A1 responses [1 µM 5'-N-ethylcarboxyamidoadenosine (NECA)] were examined pre- and post-carbachol (10 µM, CCh) application with 10 µM PIP2 in the patch pipette. NECA was applied for 5 s and carbachol for 30 s. A similar magnitude of current was induced through both A1 and M3 receptors, indicating good receptor expression (first NECA-induced current 73.6 ± 22.6 pA/pF, +CCh-induced current 61.4 ± 18.0 pA/pF, second NECA-induced current 62.6 ± 15.0 pA/pF, n = 8, P > 0.05). Cells were clamped at –60 mV and peak currents were subtracted from basal currents immediately before agonist application to calculate induced current density. A typical example is shown in (i). NECA activation of A1 receptors gives characteristic "rebound" activation of the channel on agonist wash out (34). M3 receptor activation inhibits basal channel activity but a second A1 response is seen. The time between the first and second NECA application was 5 min. The carbachol was applied 1 min after full reversal of channel potentiation after the first NECA application. Dashed line indicates zero current. B: bar chart summarizing the agonist induced current for experiments described in A, n = 8.

 
These data and observations thus suggest dual regulation of the channel: PKC clearly inhibits the channels, whereas phosphoinositide hydrolysis/turnover is involved in recovery of the channel from inhibition. In whole cell mode, both PIP2 depletion and PKC play a role in channel regulation (the former likely due to dialysis of PIP2 metabolites out of the cell into the pipette) because PKC inhibitors partially reduce the extent of M3-mediated inhibition (~60%; Ref. 27) and added PIP2 (in the pipette) partially prevents M3 inhibition (~50%). Under perforated-patch conditions, the effects of M3 stimulation are completely abolished by staurosporine, suggesting that PKC is mainly responsible for channel inhibition. Recovery from channel inhibition seems to be mediated by PIP2 on the basis of the effects of wortmannin.

We were interested in elucidating whether a specific isoform of PKC may mediate channel inhibition. Because there are very few (if any) pharmacological agents that are isoform-specific, we made several molecular tools to identify which isoform(s) may play a role. Although constitutively active and kinase-dead mutants have been previously employed by several investigators to assess PKC function, evidence exists that these mutants lose their isotype-specific features observed for wild-type PKC isotypes (9, 14). However, ectopic expression of wild-type PKC isotypes has been associated with a range of specific biological inputs, suggesting that this a more useful approach to monitor isotype-specific function (2). We generated a series of myc-tagged PKC isotypes and analyzed their effects on whole cell Kir3.1/3.2A currents measured in the HKIR3.1/3.2A cell line. These experiments were done in the presence of co-expressed G{beta}1{gamma}2 to elevate basal currents (basal: 49.7 ± 4.4 pA/pF, n = 97; +{beta}1{gamma}2: 241.3 ± 33.9 pA/pF, n = 64, P < 0.001). Both PKC-{beta}I and PKC-{delta} significantly reduced current density (PKC-{beta}I: 91.2 ± 19.8 pA/pF, n = 12, P < 0.05; PKC-{delta}: 95.1 ± 25.2 pA/pF, n = 15, P < 0.05) compared with control cells, whereas PKC-{alpha}, -{gamma}, -{epsilon}, -{eta}, and -{zeta} had no significant effects (Fig. 3B). Western blot analysis using an anti-myc antibody was used to confirm expression of these constructs, but it also revealed differences in expression levels (Fig. 3A). It is apparent from the electrophysiological data that PKC-{beta}I and PKC-{delta} had similar magnitudes of effects on current density even though they had different expression levels, as revealed by the Western blot in Fig. 3A. We thus titrated PKC-{beta}I against PKC-{delta} by reducing the amounts of PKC-{beta}I construct transfected into the cells (50, 100, and 200 ng) and measuring the expression levels using the myc tag. We found similar expression levels using 100 ng PKC-{beta}I cDNA and 1,000 ng PKC-{delta} cDNA for transfection (Fig. 4A). These amounts were then used to measure the effect on current density in the G{beta}1{gamma}2-expressing HKIR3.1/3.2A cells as described above. PKC-{beta}I had no significant effects on current density (130.1 ± 32.5 pA/pF, n = 19; P > 0.05) compared with control cells, whereas PKC-{delta} significantly reduced it (81.3 ± 16.7 pA/pF, n = 22, P < 0.01; Fig. 4B). The capacity of PKC-{delta} to reduce current density was confirmed using a different PKC-{delta} construct, in which expression was driven by a cytomegalovirus promoter. Again, PKC-{delta} expression significantly reduced current density (69.0 ± 15.4 pA/pF, n = 11, P < 0.05).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 3. Inhibition of Kir3.1/3.2A currents by isotypes of PKC. A: Western blot showing the expression of myc-tagged PKC isotypes (500 ng) transiently transfected into the HKIR3.1/3.2A stable cell line. Numbers to the left of the blot indicate molecular weight in kilodaltons. B: bar chart summarizing the effects of myc-tagged PKC isoforms on Kir3.1/3.2A current density (measured at –60 mV) in the presence of cotransfected G{beta}1{gamma}2-subunits. The open bar refers to basal currents measured in the HKIR3.1/3.2 cell line ("Ctrl"), the solid bar represents enhanced HKIR3.1/3.2 current density after the exogenous expression of G{beta}1{gamma}2 dimers ("+{beta}1{gamma}2") and gray bars indicate current density in the presence of G{beta}1{gamma}2 and each of the PKC isotypes, as indicated. Numbers in parentheses indicate the number of cells recorded.

 


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. PKC-{delta} specifically inhibits Kir3.1/3.2A channels. A: Western blot shows the expression of different amounts of myc-tagged PKC-{beta}I (50, 100, and 200 ng) and PKC-{delta} (1,000 ng) and indicates that equivalent expression levels of PKC-{beta}I and PKC-{delta} are obtained using 100 and 1,000 ng, respectively. B: similarly to Fig. 3B, 100-ng PKC-{beta}I and 1,000-ng PKC-{delta} were transfected into the HKIR3.1/3.2 cell line in the presence of G{beta}1{gamma}2 and current density measured. PKC-{delta} significantly reduced current density, whereas PKC-{beta}I had no significant effects.

 
HEK-293 cells express PKC-{alpha}, {beta}I, {beta}II, and {delta} (27). We next assessed the activation of endogenous PKC-{delta} by M3 muscarinic receptors in the HKIR3.1/3.2A/M3 stable cell line using a translocation assay. Figure 5 shows that a substantial increase in membrane association occurred after stimulation of the M3 receptor. This experiment suggests that the M3 receptors engage the endogenous pool of PKC-{delta} in these cells, directing it to the plasma membrane in the vicinity of the channel. These experiments were complemented by real time imaging studies demonstrating translocation of PKC in living HEK-293 cells. We used COOH-terminal GFP-tagged PKC-{delta} and PKC-{epsilon} (used as a negative control) constructs transiently transfected into the HKIR3.1/3.2A/M3 stable line to investigate their translocation in response to direct PKC stimulation using PMA or via receptor-mediated means with the use of carbachol. As expected, PMA resulted in an irreversible translocation of both isoforms to the plasma membrane (data not shown), whereas carbachol caused only the translocation of PKC-{delta} but not PKC-{epsilon} to the plasma membrane (Fig. 6). Translocation of PKC-{delta} was reversible on removal of carbachol and was observed at both 25°C and 35°C.



View larger version (32K):
[in this window]
[in a new window]
  		Fig. 5. Translocation of PKC-{delta} after stimulation of the M3 receptor. The translocation of PKC-{delta} was investigated in the HKIR3.1/3.2A/M3 cell line in response to either stimulation of the M3 receptor with CCh (10 µM) or to phorbol ester (PMA; 100 nM). An example of an immunoblot is shown in (A) and the bar chart summarizing the data, which was quantified by gel-scanning densitometry (see MATERIALS AND METHODS), is shown in (B). Open bars indicate fraction of PKC-{delta} at the cytosol (C) and solid bars indicate PKC-{delta} at the membrane (M).

 


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 6. Green fluorescent protein (GFP)-tagged PKC-{delta} translocation in response to muscarinic M3 receptor stimulation. Fluorescent images of HKIR3.1/3.2A/M3 cells expressing {epsilon}-PKC-GFP (A) and {delta}-PKC-GFP (B) after 0-, 2-, or 3-min application of CCh (10 µM). M3-receptor activation causes selective reversible translocation of {delta}-PKC-GFP. Line profiles for {delta}-PKC-GFP are shown beneath the appropriate time points. C: bar chart indicating significant drop in fluorescence (vs. control levels) in region of interest (ROI) cytoplasmic region (boxed region) for {delta}-PKC-GFP (P < 0.001, n = 5), but not {epsilon}-PKC-GFP (n = 5). D: line plot showing time course of change in fluorescence intensity in ROI for {delta}-PKC-GFP example in (B).

 
We further investigated the potential involvement of PKC-{delta} in Kir3.1/3.2A channel inhibition using dominant negative inhibition of the kinase. Isotype-specific inhibition may be achieved on the basis of unique domains/motifs in individual isotypes (8). For example, the regulatory domain or isolated C2 domain of PKC-{delta} inhibits cellular responses mediated by this isotype (29, 41). Figure 7 shows that the regulatory domain of PKC-{delta}, comprising C1 and C2 modules, as well as the C2 domain of PKC-{delta} on its own, both significantly attenuated the M3-mediated inhibition (control: 77.4 ± 2.7%, n = 24; regulatory: 47.5 ± 5.5%, n = 13, P < 0.001; C2-like: 56.2 ± 6.0%; n = 11, P < 0.001). The magnitude of this effect is similar to that observed in our previous studies using PKC inhibitors, which do not discriminate between PKC isoforms. Therefore, it appears that PKC-{delta} mediates the effect of M3 receptor stimulation on the current measured. Transient transfection of PKC-{delta} also reduced A1-mediated currents, whereas PKC-{beta} (at equivalent expression levels) had no significant effects. Data are summarized in Fig. 8. The PKC-{delta} effect is similar to that observed for M3 receptor-mediated attenuation of the A1-induced current that we have previously described (27) and suggests that PKC-{delta} is an important mediator of the receptor response.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7. The C2-like domain of PKC-{delta} attenuates M3 receptor-mediated channel inhibition. A: representative examples of the effects of M3 receptor stimulation on Kir3.1/3.2A current recorded from the HKIR3.1/3.2A/M3 stable cell line voltage clamped at a holding potential of –60 mV. CCh (10 µM) was applied as indicated by the bar, the dotted lines indicate zero current, and the dashed lines indicate basal current before receptor stimulation, as annotated. Currents were initially potentiated by carbachol but then decreased to a value substantially lower than that before receptor stimulation. We measured the percent inhibition of current before agonist application; this is indicated on each trace. Top, control response in HKIR3.1/3.2/M3 cells; middle, in the presence of the regulatory domain of PKC-{delta} (+PKC-{delta} Reg); and bottom, in the presence of the PKC-{delta} C2-like domain. B: we measured inhibition of current density in the HKIR3.1/3.2A/M3 cell line in response to 10 µM CCh in the absence ("M3 Ctrl") and presence of either the regulatory domain ("+PKC-{delta} Reg") or the C2-like domain ("+PKC-{delta} C2") of PKC-{delta}. *P < 0.001.

 


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 8. Expression of PKC-{delta} attenuates A1 receptor-mediated activation of Kir3.1/3.2A channels. A: representative example of the effects of A1 receptor stimulation on Kir3.1/3.2A currents in the HKIR3.1/3.2/A1 cell line in the absence (top) and presence of myc-tagged PKC-{beta}I (middle), or myc-tagged PKC-{delta} (bottom). We used 100 ng of PKC-{beta}I and 1,000 ng of PKC-{delta} because these had equivalent expression levels. Currents were elicited by holding cells at 0 mV and stepping to potentials between –100 and +50 mV in 10 mV increments for 100 ms. Current traces were recorded before ("Control"), during ("+NECA") and after receptor stimulation ("Wash"). B: bar chart summarizing the effects of PKC-{beta}I and PKC-{delta} on NECA-induced currents in the HKIR3.1/3.2/A1 cell line. Open bar ("Ctrl") indicates NECA-induced currents under control conditions, whereas the solid bar indicates NECA-induced currents in the presence of PKC-{beta}I and the gray bar in the presence of PKC-{delta}. *P < 0.05.

 
Thus our data supports a role for PKC-{delta} in Kir3.1/3.2A channel inhibition but the exact mechanism by which it acts remains unclear. One possibility is that PKC phosphorylates the channel complex thus inhibiting it directly, as observed with Kir3.1/3.4 channels by Mao et al. (30). We performed a series of experiments using cell-attached and excised patches to elucidate the mechanism of action of PKC. We first confirmed that the M3-mediated inhibition was via a diffusible messenger by making cell-attached recordings of Kir3.1/3.2A channels after stimulation of stably coexpressed M3 receptor. NPo channel activity was significantly and irreversibly decreased by carbachol (control, 0.0168 ± 0.008; +CCh, 0.0049 ± 0.002; wash, 0.0006 ± 0.0001; n = 3, P < 0.05; Fig. 9A), suggesting that the mechanism responsible for channel inhibition by Gq/11-coupled receptors, at least in part, involves a cytosolic diffusible messenger.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 9. Single channel activity of Kir3.1/3.2A channels. A: cell-attached recordings in HKIR3.1/3.2A/M3 cells demonstrates a reduction in channel activity after M3 receptor activation (10 µM CCh, 30 s), indicating the involvement of a diffusible cytosolic messenger. See RESULTS for data (n = 3). B: channel activity was recorded for 30-s sweeps at test potentials between –60 and +60 mV to examine for rectification. Channel openings were characteristically brief with a mean open time of 1–1.5 ms. Current-voltage relationships demonstrated strong inward rectification with a unitary conductance of 32 ± 2 pS (n = 8). Vm, membrane potential.

 
To further investigate the role of PKC in mediating a direct inhibition of Kir3.1/3.2A channels we used purified PKC isolated from rat brain (see MATERIALS AND METHODS) and bath applied this to inside-out patches. We first characterized the single channel properties of the HKIR3.1/3.2/A1 cell line with 1 µM adenosine in the pipette, which we have shown to maximally activate whole cell Kir3.1/3.2A currents (3). Channel mean open times were characteristically brief (~1–2 ms) with a mean open time of 32 ± 2 pS (Fig. 9B) in accordance with previous findings (15, 21). Channel activity was sensitive to GTP because removal of GTP led to rapid rundown in activity, which could be restored by subsequent replenishment of GTP.

To ensure that our preparation of rat brain PKC was clean and functional, we performed several control experiments: there were no effects of vehicle on channel activity, PKC activity required the presence of Mg2+ and ATP, and boiling the preparation to inactivate it prevented its effects (data not shown). We also confirmed our results with a commercially available preparation (Calbiochem). Thus PKC (~1.6 U/ml) rapidly (1–2 s) and significantly reduced channel opening (control, 0.554 ± 0.06; +PKC, 0.084 ± 0.02, n = 6, P < 0.001), which recovered on removal of PKC (wash, 0.518 ± 0.05; n = 6). In the presence of the phosphatase inhibitor okadaic acid (OA; 10 µM), the reversibility of the effects of PKC on channel activity was prevented (Control, 0.455 ± 0.08; +PKC, 0.008 ± 0.004; +OA, 0.06 ± 0.008; and wash 0.413 ± 0.04; n = 5, P < 0.001, Fig. 10A), suggesting a mechanism involving phosphorylation and subsequent dephosphorylation.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 10. Kir3.1/3.2A channels are inhibited by PKC channel activity was recorded in HKIR3.1/3.2A/A1 cell line using the inside-out patch configuration. Solutions were supplemented with 2 mM ATP and 100 µM GTP. A: example trace of PKC-mediated inhibition of Kir3.1/3.2A channel activity. After recording control activity, PKC (~1.6 U/ml) was applied and channel activity was rapidly inhibited. Removal of PKC and application of PP1/PP2A inibitor okadaic acid (OA; 1 µM) prevented full recovery of activity. On wash channel activity rapidly returned to control levels (see RESULTS for data, n = 5). Arrows indicate time point of applications. Maximum channel activity was achieved through inclusion of 1 µM NECA in pipette. B: example trace showing that application of 10 nM His6-{beta}{gamma} had no effect on PKC-mediated inhibition or recovery in the presence of OA. No agonist was present in the pipette. C,i: accumulated data (n = 3) for experiments conducted in B showing significant reductions of channel activity in the presence of His6-{beta}{gamma} (ii).

 
It has been proposed that Kir3 phosphorylation and binding of G{beta}{gamma} are mutually exclusive (31). Using whole cell recordings, we previously showed that M3-mediated channel inhibition can still occur in the presence of excess G{beta}1{gamma}2 (27) and we further confirm this at the single channel level in this study using purified His6-tagged G{beta}1{gamma}2 (see MATERIALS AND METHODS). 10 nM purified protein (10 nM G{beta}1{gamma}2 near maximally activates KAch currents in atrial cells; 20) was applied to inside-out patches from HKIR3.1/3.2A/A1 cells (in the absence of agonist), which led to a large increase in NPo (control, 0.004 ± 0.001; + His6-G{beta}1{gamma}2, 0.223 ± 0.024, n = 3). Application of purified PKC significantly reduced this activity (0.03 ± 0.007, P < 0.001) and the channels remained inhibited when purified His6-G{beta}1{gamma}2 was then added (+PKC/His6-G{beta}1{gamma}2, 0.003 ± 0.001, P < 0.001). The effects of PKC were reversible: however, channel activity remained significantly depressed after removal of PKC and the addition of OA to prevent phosphatase action (+OA/His6-G{beta}1{gamma}2, 0.053 ± 0.007). Single channel activity was rapidly restored on washing out OA (0.499 ± 0.070, P < 0.001). A typical example is shown in Fig. 10B and data are summarized in Fig. 10C.

It has recently been shown by Mao et al. (30) that single point mutations introduced into Kir3.1 and Kir3.4 subunits (S185A and S191A, respectively) prevented PMA and Gq/11-receptor-mediated inhibition of the Kir3.1/3.4 channel. We generated the equivalent mutations in Kir3.1 and Kir3.2C (S185A and S178A, respectively): the wild-type heteromeric channel we have found to behave similarly at the whole cell level to wild-type Kir3.1/3.2A (data not shown). The double-mutant channel Kir3.1 (S185A)/Kir3.2C (S178A) carried currents which were robust, showed strong inward rectification, and which were inhibited by 100 nM PMA (data not shown). We then examined the modulation of these channels in response to M3 receptor stimulation. We found that carbachol still significantly and irreversibly inhibited the currents (40 ± 2% inhibition) similar to that observed with wild-type channels (49 ± 2% inhibition; Fig. 11, A and B); furthermore, purified PKC still inhibited channel activity when applied to inside-out patches (control, 0.430 ± 0.10; +PKC, 0.086 ± 0.040; wash, 0.346 ± 0.092, P < 0.05, n = 4; Fig. 11C). Therefore, these mutations had no effect on the ability of PKC (activated indirectly via a Gq/11-coupled receptor, directly via a phorbol ester or by using purified PKC itself) to inhibit Kir3.1/3.2C channels. It would seem that the phosphorylation sites identified by Mao et al. (30) are important specifically for Kir3.1+3.4 channels because in our hands the equivalent mutations clearly had no effects on M3-mediated inhibition of Kir3.1/3.2 channel.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 11. Mutant Kir3.1S185A/Kir3.2C-S178A is inhibited by PKC and Gq/11-coupled M3 receptors. A: representative traces showing current voltage relationships in cells expressing Kir3.1S185A/Kir3.2C-S178A channels before (i) and after (ii) M3 receptor activation. Current-voltage relationships are reduced to a similar extent as seen with the wild-type channel (n = 10, see RESULTS for data comparison). The inhibitory profile of M3 receptors after agonist application (CCh, 30 s) is the same with Kir3.1S185A/Kir3.2C-S178A channels as with the wild-type (ii). Control currents are indicated and the dashed line represents zero current (n = 10). B: summarized data for the reduction in Kir3.1S185A/Kir3.2C-S178A currents after M3 receptor stimulation in A. C,i: single channel activity is reduced on application of PKC to inside-out patches taken from cells expressing Kir3.1S185A/Kir3.2C-S178A channels. The use of low resistance pipettes (~4 M{Omega}) allowed recording of high channel activity. ii, bar chart summarizing data from experiments in C (n = 4). D: autoradiograph showing maltose binding protein (MBP) protein, MBP+Kir3.1 COOH termini, and MBP+Kir3.2A COOH termini in the absence (–) and presence (+) of 32P-ATP. Marker sizes are indicated in kilodaltons.

 
We examined whether PKC phosphorylates the Kir3 subunits by fusing the cytoplasmic COOH terminals of Kir3.1 and Kir3.2A to MBP and incubating with 32P-ATP and the catalytic subunit of PKC, followed by SDS-PAGE. Incorporation of radioactive phosphate into the COOH terminal domains was determined by autoradiography, as we have previously described (37). Figure 11D clearly shows that the COOH terminus of Kir3.1 is heavily phosphorylated, contrasting with the COOH terminus of Kir3.2A, which is not. Similarly, the Kir3.1 subunit is heavily phosphorylated in native atrial Kir3.1/3.4 channels (31).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Classically, the Kir3 family of inwardly rectifying K+ channels is regulated by G proteins, being activated by Gi/o-coupled receptors but not Gs-coupled receptors (51). This has been demonstrated using both cloned and native channels in a variety of systems. Several groups have also observed that both cloned and native channels are inhibited by Gq/11-coupled receptors and two mechanisms have been proposed to account for this inhibitory phenomenon: one is via the metabolism of membrane PIP2 (22, 28, 33) and the other is via activation of PKC (4, 17, 27, 30, 34, 42, 44, 48). We have shown previously that Gq/11-coupled receptor-mediated inhibition of the cloned neuronal Kir3.1/3.2A channel involves a Ca2+-independent isoform of PKC (27). In this study, we now present further evidence to support the role of PKC, likely the PKC-{delta} isotype, in mediating modulation of Kir3.1/3.2A channels. This modulation is twofold: a direct phosphorylation of the channel by PKC, which inhibits activity and a second more complex form of modulation that makes the channel more sensitive to changes in membrane phospholipid levels, possibly in a similar manner to that seen with KCNQ channels (13).

The involvement of PKC in this pathway is supported by our observation that direct pharmacological activation of PKC by phorbol esters and diacylglycerol analogues and indirect activation by Gq/11-coupled receptors leads to irreversible channel inhibition. This is further supported by our current findings that purified PKC protein inhibits channel activity when applied to excised patches, similar to the findings of Mao et al. (30) and Nikolov and Ivanova-Nikolova (34) with recombinant and native KAch channels, respectively. We found that channel activity rapidly recovered on removal of PKC indicating a close association of protein phosphatases with the channel, as suggested by Nikolov and Ivanova-Nikolova (34). This was also alluded to by Takano et al. (47), who demonstrated that the PP1/2A inhibitor OA, prevented recovery of channel activity following activation of Gq/11-coupled receptors in neurones. In a similar fashion, we also found that OA prevented the recovery of channel activity after PKC activation.

It has been proposed that PKC phosphorylates a serine residue on both Kir3.1 and Kir3.4 subunits because mutation of these residues to alanine in both subunits prevented the PKC mediated inhibitory phenomenon on the heteromeric channel (30). We introduced the equivalent mutations into Kir3.1 and Kir3.2C subunits and examined the properties of this heteromeric double mutant channel in response to 1) PMA treatment, 2) Gq/11 receptor stimulation, and 3) application of PKC to inside-out patches. Interestingly, all three mechanisms for investigating the effects of PKC on channel activity resulted in a similarly significant decrease in Kir3.1(S185A)/Kir3.2C(S178A) channel currents compared with wild-type Kir3.1/3.2C channels. This would seem to imply that the profile of PKC-phosphorylation of Kir3.1/3.2 heteromers is far more complex than that of Kir3.1/3.4, supported by the fact that Kir3.2 contains 26 phosphorylation sites compared with 19 in Kir3.4 (NetPhos predicted). Our biochemical data clearly shows that Kir3.1 is heavily phosphorylated by PKC, lending support to the notion that PKC can modify the channel on multiple sites. Perhaps with Kir3.1/3.2 channels, multiple sites are involved in a stepwise fashion. In addition, the environment of a Xenopus oocyte as used by Mao et al. (30) may differ substantially than the mammalian cells used in the current study.

This study has provided further evidence reinforcing our previous hypothesis that a Ca2+-independent PKC isotype is responsible for the inhibitory phenomenon (27). PKC-{delta} would appear to be the strongest candidate because, after controlling for levels of expression, PKC-{delta} was the only isotype, which caused a significant reduction in both basal and agonist-induced Kir3.1/Kir3.2A currents. Conversely, disruption of endogenous PKC-{delta} activity through expression of dominant negative PKC domains significantly reduced the Gq/11-mediated channel inhibition. Translocation of GFP-tagged PKC-{delta}, but not PKC-{epsilon}, in our HKIR3.1/3.2A/M3 stable cell line after receptor stimulation provides further evidence for the selective activation and recruitment of PKC-{delta} to the membrane, which can then act to inhibit Kir3.1/3.2 channel activity. The inhibition of the M3 receptor response by the regulatory domain of PKC-{delta} does not itself indicate that PKC-{delta} is the only isotype involved in the response. The regulatory domain contains the C1 module, which binds endogenous diacylglycerol and on this basis would inhibit most PKC isotypes, including classic PKCs. However, the fact that the PKC-{delta} C2 module on its own antagonizes the M3 receptor response suggests that PKC-{delta} is the most likely isotype involved. This module represents a unique fold within the PKC family (displaying only significant similarity with PKC-{theta}, which is not expressed in these cells; Ref. 27) and as such would have a specific inhibitory effect (35). The mechanism by which this inhibition occurs is not clear. PKC-{delta}C2 has been identified as a protein interaction module (8) and has inhibitory effects on PKC-{delta} activity in other cellular contexts (29). It may therefore reduce the interaction of PKC-{delta} with essential binding partners, e.g., receptor for activated C kinase (34) or A-kinase anchoring protein (13) so that channel modulation does not occur. The identification of these binding partners, and the biochemical characterization of the interaction with respect to isotype preference, will provide further insight into the mechanism of regulation of this class of channel.

Clearly, the native cellular environment is organized and expression is controlled, and therefore PKC recruitment will depend on stimulus, tissue expression, and subcellular microdomains (11, 32). Indeed, evidence has been presented that Ca2+-dependent PKC isotypes are recruited to atrial membranes after {alpha}-adrenoceptor stimulation and can inhibit KAch currents (34). Therefore, it is tempting to speculate that endogenous Kir3 currents may be inhibited through pathways that stimulate different isoforms of PKC. In our single channel studies, PKC-mediated inhibition reversed rapidly on removal of the enzyme. Consequently, it is unlikely that PKC is causing the prolonged inhibition of channel activity after M3 receptor activation in whole cell conditions. Therefore, it is probable that the loss of phospholipid from the membrane is the cause of this.

Du and co-workers (12) have proposed that Kir3 channels have a comparatively low affinity (vs. other potassium channel family members) for PIP2 and thus are susceptible to PKC-mediated inhibition (as seen through phorbol ester stimulation; 12). Our experiments examining M3-modulation of currents under whole cell and perforated-patch conditions may help begin to explain the conflicting arguments for the mediator responsible for Kir3 current inhibition. For example, when the cellular environment remained largely intact (perforated patch) PKC activity was critical for channel inhibition. However, under whole cell conditions, although to a lesser extent, channel inhibition still occurred after PKC inhibition both pharmacologically and biochemically. The prolonged inhibition was manifested probably through loss of phospholipid content as supplementation of PIP2 restored and rescued channel activity. Equally, if the native cellular environment has a low phospholipid content, then it is likely that channel inhibition could manifest as apparently only a phospholipid-dependent mechanism after Gq/11-receptor activation (13). Conversely, on the basis of our perforated-patch experiments and inhibition of PKC activity, it is tempting to speculate that despite the comparitively low PIP2 affinity, the affinity of the channel for PIP2 may be sufficient to withstand physiological changes (through Gq/11-receptor mediated activity) in membrane phospholipid content in the absence of PKC modulation under conditions of high local concentrations of phospholipid.

It is our hypothesis that heteromeric channels are modulated primarily by PKC, and secondarily by PIP2 after activation of Gq/11-coupled receptors in HEK-293 cells. Thus we present a unifying mechanism for inhibitory regulation of Kir3.1/3.2 channels involving PKC-{delta} and PIP2 after Gq/11-receptor stimulation.


   ACKNOWLEDGMENTS
 
This work is supported by the Royal Society, the Wellcome Trust, and the British Heart Foundation. The green fluorescent protein-tagged PKC-{delta} and PKC-{epsilon} constructs were a kind gift from N. Saito (Kobe University, Japan).

Present address for L. V. Dekker: Ionix Pharmaceuticals, 418 Cambridge Science Park, Milton Rd., Cambridge CB4 0PA, UK.

Present address for J. L. Leaney: Ion Channel Pharmacology Group, Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Rd., Sandwich, Kent, CT13 9NJ, UK.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. G. Brown, Dept. of Medicine, UCL, The Rayne Bldg., 5 University St., London WC1E 6JJ, UK (e-mail: sean.brown{at}ucl.ac.uk)

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
 REFERENCES
 
1. Allen BG, Andrea JE, and Walsh MP. Identification and characterization of protein kinase C{zeta}-immunoreactive proteins. J Biol Chem 269: 29288–29298, 1994.[Abstract/Free Full Text]

2. Akita Y, Kawasaki H, Ohno S, Suzuki K, and Kawashima S. Involvement of protein kinase C{epsilon} in thyrotropin-releasing hormone-stimulated phosphorylation of myristoylated alanine-rich C kinase substrate in rat pituitary clonal cells. Electrophoresis 21: 452–459, 2000.[CrossRef][Web of Science][Medline]

3. Benians A, Leaney J, and Tinker A. Agonist unbinding from receptor dictates the nature of deactivation kinetics of G protein-gated K+ channels. Proc Natl Acad Sci USA 100: 6239–6244, 2003.[Abstract/Free Full Text]

4. Chen Y and Yu L. Differential regulation by cAMP-dependent protein kinase and protein kinase C of the µ opiod receptor coupling to a G protein-activated K+ channel. J Biol Chem 269: 7839–7842, 1994.[Abstract/Free Full Text]

5. Cho H, Nam GB, Lee SH, Earm YE, and Ho WK. Phosphatidylinositol 4,5-bisphosphate is acting as a signal molecule in {alpha}1-adrenergic pathway via the modulation of acetylcholine-activated K+ channels in mouse atrial myocytes. J Biol Chem 276: 159–164, 2001.[Abstract/Free Full Text]

6. Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, Francke U, and Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science 233: 859–866, 1986.[Abstract/Free Full Text]

7. Coussens L, Rhee L, Parker PJ, and Ullrich A. Alternative splicing increases the diversity of the human protein kinase C family. DNA (NY) 6: 389–394, 1987.

8. Dekker LV and Parker PJ. Regulated binding of the protein kinase C substrate GAP-43 to the V0/C2 region of protein kinase C-{delta}. J Biol Chem 272: 12747–12753, 1997.[Abstract/Free Full Text]

9. Dekker LV, Parker PJ, and McIntyre P. Biochemical properties of rat protein kinase C-{eta} expressed in COS cells. FEBS Lett 312: 195–199, 1992.[CrossRef][Web of Science][Medline]

10. Dekker LV, McIntyre P, and Parker PJ. Mutagenesis of the regulatory domain of rat protein kinase C-{eta}. A molecular basis for restricted histone kinase activity. J Biol Chem 268: 19498–19504, 1993.[Abstract/Free Full Text]

11. Delmas P, Crest M, and Brown DA. Functional organisation of PLC signaling microdomains in neurons. Trends Neurosci 27: 41–47, 2004.[CrossRef][Web of Science][Medline]

12. Du X, Zhang H, Lopes C, Mirshahi T, Rohacs T, and Logothetis DE. Characteristic interactions with phosphatidylinositol 4,5 bisphosphate determine regulation of Kir channels by diverse modulators. J Biol Chem 279: 37271–37281, 2004.[Abstract/Free Full Text]

13. Filipov AK, Fernandez-Fernandez JM, Marsh SJ, Simon J, Barnard EA, and Brown DA. Activation and inhibition of neuronal G protein-gated inwardly rectifying K+ channels by P2Y nucleotide receptors. Mol Pharmacol 66: 468–477, 2004.[Abstract/Free Full Text]

14. Garcia-Paramio P, Cabrerizo Y., Bornancin F, and Parker PJ. The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation. J Biol Chem 333: 631–636, 1998.

15. Grigg JJ, Kozasa T, Nakajima Y, and Nakajima S. Single channel properties of a G-protein gated inward rectifier potassium channel in brain neurons. J Neurophysiol 75: 318–328, 1996.[Abstract/Free Full Text]

16. Hoshi N, Zhang J, Omaki M, Takeuchi T, Yokoyama S, Wanaverbecq N, Langberg J, Yoneda Y, Scott JD, Brown DA, and Higashida H. AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists. Nat Neurosci 6: 564–571, 2003.[CrossRef][Web of Science][Medline]

17. Hil JJ and Peralta EG. Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled M1 muscarinic acetylcholine receptor. J Biol Chem 276: 5505–5510, 2001.[Abstract/Free Full Text]

18. Ho IHM and Murrell-Lagnado RD. Molecular determinants for sodium-dependent activation of G protein-gated K+ channels. J Biol Chem 274: 8639–8648, 1999.[Abstract/Free Full Text]

19. Huang CL, Feng S, and Hilgemann DW. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gbetagamma. Nature 19: 803–806, 1998.

20. Ito H, Tung RT, Sugimoto T, Kobayashi I, Takahashi K, Katada T, Ui M, and Kurachi Y. On the mechanism of basal and agonist-induced activation of the G-protein-gated muscarinic K+ channel in atrial myocytes of the guinea-pig heart. J Gen Physiol 3: 517–513, 1992.

21. Jelacic TM, Sims SM, and Clapham DE. Functional expression and characterization of G-protein-gated inwardly rectifying K+ channels obtaining GIRK3. J Membr Biol 169: 123–129, 1999.[CrossRef][Web of Science][Medline]

22. Kobrinksy E, Mirshahi T, Zhang H, Jin T, and Logothetis DE. Receptor-mediated hydrolysis of plasma membrane PIP2 leads to K+-current desensitization. Nat Cell Biol 2: 507–514, 2000.[CrossRef][Web of Science][Medline]

23. Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, and Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281: 2042–2045, 1998.[Abstract/Free Full Text]

24. Leaney JL. Contribution of Kir3.1, Kir3.2, and Kir3.2 subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons. Eur J Neurosci 18: 2110–2118, 2004.

25. Leaney JL, Milligan G, and Tinker A. The G protein {alpha} subunit has a key role in determining the specificity of coupling to, but not the activation of, G protein-gated inwardly rectifying K+ channels. J Biol Chem 275: 921–929, 2000.[Abstract/Free Full Text]

26. Leaney JL and Tinker A. The role of members of the pertussis toxin-sensitive family of G proteins in coupling receptors to the activation of the G protein-gated inwardly rectifying potassium channel. Proc Natl Acad Sci USA 97: 5651–5656, 2000.[Abstract/Free Full Text]

27. Leaney JL, Dekker LV, and Tinker A. Regulation of a G-protein gated inwardly rectifying channel by a Ca2+-independent protein kinase C. J Physiol 534: 367–379, 2001.[Abstract/Free Full Text]

28. Lei Q, Talley E, and Bayliss DA. Receptor-mediated inhibitoin of G protein coupled inwardly rectifying potassium channels involves G{alpha}q family subunits, phospholipase C and a readily diffusible messenger. J Biol Chem 276: 16720–16730, 2001.[Abstract/Free Full Text]

29. Lopez-Lluch G, Bird MM, Canas B, Godovac-Zimmerman J, Ridley A, Segal AW, and Dekker LV. Protein kinase C-{delta} C2-like domain is a binding site for actin and enables actin redistribution in neutrophils. Biochem J 357: 39–47, 2001.[CrossRef][Web of Science][Medline]

30. Mao J, Wang X, Chen F, Wang R, Rojas A, Shi Y, Piao H, and Jiang C. Molecular basis for the inhibition of G protein-coupled inward rectifier K+ channels by protein kinase C. Proc Natl Acad Sci USA 101: 1087–1092, 2004.[Abstract/Free Full Text]

31. Medina I, Krapivinsky G, Arnold S, Kovoor P, Krapivinsky L, and Clapham DE. A switch mechanism for G{beta}{gamma} activation of I (KACh). J Biol Chem 275: 29709–29716, 2000.[Abstract/Free Full Text]

32. Mellor H and Parker PJ. The extended protein kinase C superfamily. Biochem J 332: 281–292, 1998.

33. Meyer T, Wellner-Kienitz M, Biewald A, Bender K, Eickel A, and Pott L. Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes. J Biol Chem 276: 5650–5658, 2001.[Abstract/Free Full Text]

34. Nikolov EN and Ivanova-Nikolova TT. Coordination of membrane excitability through a GIRK1 signaling complex in the atria. J Biol Chem 279: 23630–23636, 2004.[Abstract/Free Full Text]

35. Pappa H, Murray-Rust J, Dekker LV, Parker PJ, and McDonald NQ. Crystal structure of the C2 domain from protein kinase C-{delta}. Structure 7: 885–894, 1998.[CrossRef]

36. Parker PJ, Coussens L, Totty N, Rhee L, Young S, Chen E, Stabel S, Waterfield MD, and Ullrich A. The complete primary structure of protein kinase C–the major phorbol ester receptor. Science 233: 853–859, 1986.[Abstract/Free Full Text]

37. Quinn KV, Cui Y, Giblin JP, Clapp LH, and Tinker A. Do anionic phospholipids serve as cofactors or second messengers for the regulation of activity of cloned ATP-sensitive K+ channels? Circ Res 93: 646–655, 2003.[Abstract/Free Full Text]

38. Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez-Lluhi JA, Lefowitz RJ, Bourne HR, Jan YN, and Jan LY. Activation of the cloned muscarinic potassium channel by G protein-{beta}{gamma} subunits. Nature 370: 143–146, 1994.[CrossRef][Medline]

39. Rohács 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]

40. Schaap D, Parker PJ, Bristol A, Kriz R, and Knopf J. Unique substrate specificity and regulatory properties of PKC-{epsilon}: a rationale for diversity. FEBS Lett 243: 351–357, 1989.[CrossRef][Web of Science][Medline]

41. Schuringa JJ, Dekker LV, Vellenga E, and Kruijer W. Sequential activation of Rac-1, SEK-1/MKK-4 and PKC-{delta} is required for interleukin-6 induced STAT3 Ser-727 phosphorylation and transactivation. J Biol Chem 276: 27709–27715, 2001.[Abstract/Free Full Text]

42. Sharon D, Vorobiov D, and Dascal N. Positive and negative coupling of the metabotropic glutamate receptors to a G protein-activated K+ channel, GIRK, in Xenopus oocytes. J Gen Physiol 109: 477–490, 1997.[Abstract/Free Full Text]

43. Shirai Y, Sakai N, and Saito N. Subspecies-specific targeting mechanism of protein kinase C. Jpn J Pharmacol 78: 411–417, 1998.[CrossRef][Medline]

44. Stanfield PR, Nakajima S, and Nakajima Y. Constitutively active and G-protein coupled inward rectifier K+ channels: Kir2.0 and Kir3.0 Rev Physiol. Biochem Pharmacol 145: 47–179, 2002.

45. Stevens EB, Shah BS, Pinnock RD, and Lee K. Bombesin receptors inhibit G protein-coupled inwardly rectifying K+ channels expressed in Xenopus oocytes through a protein kinase C-dependent pathway. Mol Pharmacol 55: 1020–1027, 1999.[Abstract/Free Full Text]

46. Suh BC and Hille B. Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35: 507–520, 2002.[CrossRef][Web of Science][Medline]

47. Takano K, Stanfield PR, Nakajima S, and Nakajima Y. Protein kinase C-mediated inhibition of an inward rectifier potassium channel by substance P in nucleus basalis neurons. Neuron 14: 999–1008, 1995.[CrossRef][Web of Science][Medline]

48. Towbin H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci 76: 4350–4354, 1979.[Abstract/Free Full Text]

49. Thomas AM, Giblin J, Wilson A, and Tinker A. A biochemical approach to studying the interaction of anionic phospholipids with potassium channel domains. J Physiol 557P: PC85, 2004.

50. Wickman KD, Iniguez-Lluhl JA, Davenport PA, Taussig R, Krapivinsky GB, Linder ME, Gilman AG, and Clapham DE. Recombinant G protein {beta}{gamma}-subunits activate the muscarinic-gated atrial potassium channel. Nature 368: 255–257, 1994.[CrossRef][Medline]

51. Yamada M, Inanobe A, and Kurachi Y. G protein regulation of potassium ion channels. Pharmacol Rev 50: 723–760, 1998.[Abstract/Free Full Text]

52. Zeng W, Li X, Hilgemann DW, and Huang C. Protein kinase C inhibits ROMK1 channel activity via a phsophatidylinositol 4,5-bisphosphate-dependent mechanism. J Biol Chem 278: 16825–16856, 2003.

53. 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][Web of Science][Medline]




This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
J. Luo, J. M. Busillo, and J. L. Benovic
M3 Muscarinic Acetylcholine Receptor-Mediated Signaling Is Regulated by Distinct Mechanisms
Mol. Pharmacol., August 1, 2008; 74(2): 338 - 347.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Kanchanapoo, M. Ao, R. Prasad, C. Moore, C. Kay, P. Piyachaturawat, and M. C. Rao
Role of protein kinase C-{delta} in the age-dependent secretagogue action of bile acids in mammalian colon
Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1851 - C1861.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
J. Qiu, C. Xue, M. A. Bosch, J. G. Murphy, W. Fan, O. K. Ronnekleiv, and M. J. Kelly
Serotonin 5-Hydroxytryptamine2C Receptor Signaling in Hypothalamic Proopiomelanocortin Neurons: Role in Energy Homeostasis in Females
Mol. Pharmacol., October 1, 2007; 72(4): 885 - 896.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
D. A. Brown, S. A. Hughes, S. J. Marsh, and A. Tinker
Regulation of M(Kv7.2/7.3) channels in neurons by PIP2 and products of PIP2 hydrolysis: significance for receptor-mediated inhibition
J. Physiol., August 1, 2007; 582(3): 917 - 925.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
J.-W. Sohn, A. Lim, S.-H. Lee, and W.-K. Ho
Decrease in PIP2 channel interactions is the final common mechanism involved in PKC- and arachidonic acid-mediated inhibitions of GABAB-activated K+ current
J. Physiol., August 1, 2007; 582(3): 1037 - 1046.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
J.-W. Sohn, D. Lee, H. Cho, W. Lim, H.-S. Shin, S.-H. Lee, and W.-K. Ho
Receptor-specific inhibition of GABAB-activated K+ currents by muscarinic and metabotropic glutamate receptors in immature rat hippocampus
J. Physiol., April 15, 2007; 580(2): 411 - 422.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Q. Jia, Z. Jia, Z. Zhao, B. Liu, H. Liang, and H. Zhang
Activation of Epidermal Growth Factor Receptor Inhibits KCNQ2/3 Current through Two Distinct Pathways: Membrane PtdIns(4,5)P2 Hydrolysis and Channel Phosphorylation
J. Neurosci., March 7, 2007; 27(10): 2503 - 2512.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Tai, J. B. Kuzmiski, and B. A. MacVicar
Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons.
J. Neurosci., June 7, 2006; 26(23): 6249 - 6258.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
G. E. Breitwieser
GIRK channels: hierarchy of control. Focus on "PKC-{delta} sensitizes Kir3.1/3.2 channels to changes in membrane phospholipid levels after M3 receptor activation in HEK-293 cells"
Am J Physiol Cell Physiol, September 1, 2005; 289(3): C509 - C511.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/3/C543    most recent
00025.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, S. G.
Right arrow Articles by Leaney, J. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, S. G.
Right arrow Articles by Leaney, J. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.