Published online before print April 15, 2004, doi: 10.1161/01.RES.0000128513.34817.c4
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© 2004 American Heart Association, Inc.
Cellular Biology |
Multisite Phosphorylation Mechanism for Protein Kinase A Activation of the Smooth Muscle ATP-Sensitive K+ Channel
From the British Heart Foundation Laboratories and the Department of Medicine, University College London, UK.
Correspondence to Andrew Tinker, Department of Medicine, University College London, 5 University St, London WC1E 6JJ, UK. E-mail a.tinker{at}ucl.ac.uk
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Abstract |
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The activation of ATP-sensitive K+ channels by protein kinase A in vascular smooth muscle is an important component of the action of vasodilators. In this study, we examine the molecular mechanisms of regulation of the cloned equivalent of this channel comprising the sulfonylurea receptor 2B and the inward rectifier 6.1 subunit (SUR2B/Kir6.1). Specifically, we focus on whether the channel is directly phosphorylated and the sites at which this occurs in the protein complex. We identify one site in Kir6.1 (S385) and two sites in SUR2B (T633 and S1465) using a combination of biochemical and functional assays. Our work supports a model in which multiple sites in the channel complex have to be phosphorylated before activation occurs.
Key Words: Kir6.1 • ATP-sensitive potassium channel • ion channel regulation • protein kinase A • protein phosphorylation
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Introduction |
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ATP-sensitive potassium (KATP) channels are found in a variety of tissues, and one of their roles is to allow the metabolic state of the cell to be reported at the plasma membrane.1–4 The channels exist in the cell membrane as an octomer of 4 pore-forming inward rectifier K+ (Kir6.X) subunits with 4 sulfonylurea receptor (SUR) subunits.5,6 The channels are closed by raised intracellular ATP, stimulated by MgADP, and are sensitive to K+ channel openers such as pinacidil and diazoxide. In addition, the channels are blocked by antidiabetic sulfonylureas.1–4 The differences in physiological and pharmacological properties in specific tissues are accounted for by differential subunit expression.7–9
The phosphorylation of ion channels by protein kinases is an important mechanism by which membrane excitability is regulated by cell signaling pathways.10 This is of particular physiological importance for KATP channels in vascular smooth muscle, where they control smooth muscle tone in response to hormones and neurotransmitters.11–13 In particular, endogenous vasodilators such as calcitonin gene-related peptide and adenosine have been shown to act via protein kinase A (PKA) to stimulate glibenclamide-sensitive currents in vascular smooth muscle.14–16 What is the molecular counterpart of the channel in smooth muscle? The pharmacological properties of the native channel are compatible with the known properties of SUR2B-containing channel complexes.9 Furthermore, channel activity in many native smooth muscles is absolutely dependent on nucleotide diphosphates, and this is characteristic of the behavior of Kir6.1.17–19 Finally, more direct studies of this issue with the Kir6.1 and SUR2 knockout mice and our own work in a human primary pulmonary artery smooth muscle cell line are also consistent with the native vascular channel being composed of Kir6.1/SUR2B.20–22
However, little is known of the molecular mechanisms by which protein kinases and more specifically PKA modulate the vascular KATP channel. We investigate this problem here using biochemical and electrophysiological approaches. We ask whether channel modulation occurs with the cloned channel isoforms, whether this is mediated by direct phosphorylation of the protein complex, and which residues are involved.
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Materials and Methods |
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Molecular Biology
Stable lines were generated in human embryonic kidney 293 (HEK293) cells containing either Kir6.1 with SUR2B or Kir6.2 with SUR2B, as described previously.23 The consensus site recognition program in Prosite ([R/K]-[R/K]-x-[S/T] for PKA; http://ca.expasy.org/tools/scnpsite.html) was used to identify 2 PKA consensus sites (T234, S385) in Kir6.1 and 2 PKA consensus sites in SUR2B (T633, S1465). Single point mutations were introduced into Kir6.1 and SUR2B using the QuikChange kit (Stratagene) according to the instructions of the manufacturer. Transient transfections were performed using lipofectamine (Invitrogen) or Polyfect (Qiagen), with enhanced green fluorescent protein-N1 (Clontech) as a marker for transfected cells. The cystic fibrosis transmembrane conductance regulator (CFTR) was used as previously described.24
Generation of Maltose-Binding Protein Fusions for SUR2B NBD1 and NBD2
Polymerase chain reaction (PCR) products corresponding to NBD1 (amino acids 623–957) and NBD2 (amino acids 1322–1546) of mouse SUR2B were subcloned in frame into the pMALc2x expression vector (New England Biolabs). A shortened version of the NBD1 fusion protein with and without a point mutation (amino acids 623–734, SUR2NBD1s, T633As) was also made in a similar fashion. A fusion of maltose-binding protein (MBP) with the C terminus of Kir6.1 (amino acids 179–424) was generated in a previous study.19 MBP fusions containing mutated PKA sites were generated in an identical manner using the mutant cDNA as the PCR template.
Production and Purification of MBP Fusion Proteins
Transformation, expression, and purification of fusion proteins was done as described previously.19 A small aliquot of bound fusion protein was eluted from the resin with 10 mmol/L maltose in column buffer, and the protein content was measured using a protein assay kit (Bio-Rad).
In Vitro Phosphorylation Assays
The fusion proteins bound to the amylose resin were washed 5x1 mL HEPES buffer (50 mmol/L HEPES, pH 7.4, 1 mmol/L EDTA, 10% glycerol, 5 mmol/L sodium pyrophosphate, 50 mmol/L sodium fluoride, 1 mmol/L dithiothreitol, EDTA-free complete protease inhibitor cocktail [Roche]). A total of 5 U of the catalytic subunit of PKA (PKAcat), 12 µL of 5x phosphorylation buffer (50 mmol/L HEPES, pH 7.4, 1 mmol/L ATP, 50 mmol/L MgCl2), 26 µL of HEPES buffer, 13 µL of 50 mmol/L HEPES, pH 7.4+1.5% (v/v), Triton X-100, 1 µL of 32P-
-ATP (370 MBq/mL, 110 TBq/mmol/Lol; Amersham Biosciences) was added to 10 µg of protein. The sample was incubated at 37°C for 2 hours and then washed 5 times with 1 mL HEPES buffer. The protein was eluted with 6x Laemmli gel-loading buffer and run on an SDS-PAGE gel, dried, and subjected to autoradiography. Quantitation was performed using Scion Image (Scion).
Peptide Phosphorylation Experiments
Peptides corresponding to amino acids 629–640 of SUR2B were synthesized and high-performance liquid chromatography-purified to 85% purity (Eurogentec). Two peptides were synthesized: 1 corresponding to the wild-type sequence-designated 633T (CKKHTGVQSKPI) and the other (designated 633A) containing an alanine mutation of the putative phosphorylation site (CKKHAGVQSKPI). A total of 1 mg of each peptide was coupled at a concentration of 1 mg/mL in 90 mmol/L HEPES, pH 7.2, to 500 µL of Affi-gel 10 (Bio-Rad) according to the instructions of the manufacturer. The efficiency of coupling was observed to be 100% as assessed by protein assay (Bio-Rad), and the protein concentration of a 1:1 slurry of coupled peptide was 1 mg/mL. Phosphorylation experiments were done in the manner described earlier in this section until the final wash step using 100 µg of coupled peptide per reaction. After the final wash, the coupled peptide was resuspended in 1 mL of distilled water, and the radioactivity in three 250-µL aliquots was determined by counting in 10 mL of distilled water using a Tri-Carb 2100TR liquid scintillation analyzer (Packard Biosciences).
Electrophysiology
Membrane currents were studied with the whole-cell and inside-out patch-clamp technique using an Axopatch 200B amplifier (Axon Instruments). Currents were filtered at 1 kHz and sampled at 2 kHz via a Digidata 1200 interface. Data were analyzed using Clampfit, Fetchan, and Pstat software (Axon Intruments). Single channel open probability (NPo) was calculated from 1 minute of continuous recording, generally with multiple channels present in the patch. Whole-cell patch pipettes were manufactured from borosilicate glass (OD 1.5 mm, ID 1.2 mm) using a PP-830 puller and fire polished using an MF-830 microforge (Narishige) to give pipette resistances of 2 to 3.5 mol/L
. Excised patch pipettes were manufactured in a similar way, using glass with thicker walls (OD 1.5 mm, ID 0.86 mm), with final pipette resistances of 5 to 10 mol/L. Pipette capacitance was reduced by coating tips with a parafilm/mineral oil suspension and was compensated for electronically, whereas series resistance was compensated to 70% using the amplifier. Whole-cell bath solutions (pH 7.4) contained the following (in mmol/L): 140 KCl, 5 HEPES, 1.2 MgCl2, and 2.6 CaCl2. Pipette solutions (pH 7.2) contained the following (in mmol/L): 140 KCl, 5 HEPES, 1.2 MgCl2, 10 EGTA, 1 CaCl2, 1 (Mg)ATP, and 0.5 (Na)UDP. In the whole-cell configuration, currents generally took 10 minutes after breakthrough to fully stabilize at a steady state. For excised patch recordings, bath solutions (pH 7.2) contained the following (in mmol/L): 140 KCl, 5 HEPES, 1.2 MgCl2, 10 EGTA, 1 CaCl2 [supplemented with 1 mmol/L (Mg)ATP and 0.5 (Na)UDP where indicated]. Pipette solutions (pH 7.4) contained the following (in mmol/L): 140 KCl, 5 HEPES, 1.2 MgCl2, and 2.6 CaCl2. For experiments using CFTR, bath solutions (pH 7.4) contained the following (in mmol/L): 160 NaCl, 6 KCl, 10 HEPES, 1 MgCl2, and 1.5 CaCl2. Pipette solutions (pH 7.2) contained the following (in mmol/L): 100 K aspartate, 40 KCl, 10 HEPES, 1 MgCl2, 1.9 CaCl2, 5 EGTA (free Ca2+=100 nM), and 3 (Na)ATP. Forskolin was dissolved in dimethyl sulfoxide (DMSO) and diluted so that bath concentration of DMSO did not exceed 0.1% and so control experiments with DMSO alone had no effect on currents. Currents were measured at –60 mV unless stated otherwise. Drugs were applied either using a gravity-driven perfusion system or a pipette manifold solution changer (MSC-200; Biologic).
The cAMP Assay
For the measurement of forskolin-mediated accumulation of cAMP, HEK293 cells were grown to 20% confluence in 6-well dishes. Cells were prelabeled with 5 µCi of [3H]adenine per well (in minimum essential medium) overnight at 37°C and then incubated with the phosphodiesterase inhibitor Ro20–1724 (100 µmol/L) in serum-free medium for 30 minutes at 37°C. Forskolin (10 µmol/L) was then added and incubated for 15 minutes at 37°C. Medium was then aspirated and cells washed with serum-free medium. Reactions were terminated by the addition of 2.5% perchloric acid and 0.1 mmol/L cAMP at 4°C. [3H]cAMP was isolated by sequential chromatography using Dowex 50-alumina columns. Each fraction was collected in a vial containing 5 mL of Ultima Gold MV scintillant (Packard) and counted in a liquid scintillation counter. Reactions were done in triplicate, and data are expressed as percentage conversion of [3H]ATP to [3H]cAMP.
Data Analysis
Data are expressed as mean±SEM. Either Student paired t test or 1-way ANOVA with a Bonferroni post hoc test was used as appropriate to calculate statistical significance. All data presentation and statistical analysis were performed using Origin 6.0 (Microcal) or Prism v3.0 (GraphPad).
Reagents
All compounds were from VWR International, with the exception of forskolin, 1,9-dideoxy forskolin (Calbiochem), PKAcat, (Mg)ATP, (Na)UDP, glibenclamide, diazoxide (Sigma), levcromakalim, Rp-cAMPS, and Ro20–1724 (Tocris Cookson).
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Results |
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We first established that HEK293 cells possessed the relevant cellular signaling machinery for PKA-mediated channel modulation. We have previously observed that forskolin, a direct activator of adenylate cyclase,25 was able to elevate cAMP in HEK293 cells.26 We confirmed that it was able to do so in the Kir6.1/SUR2B stable line (Figure 1A). The CFTR is a chloride channel, the activity of which is characteristically dependent on direct PKA phosphorylation of the channel protein. Application of forskolin to HEK293 cells transiently expressing CFTR led to pronounced activation of a chloride current (Figure 1B).
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) and after perfusion with 10 µmol/L forskolin (
; n=9). Currents shown are mean of 300 ms of recording taken from the middle of each 1-second voltage step. *P<0.05.We then addressed whether cloned KATP channels could be activated by PKA in the whole-cell configuration. We have described and characterized stable HEK293 cell lines expressing Kir6.1/SUR2B or Kir6.2/SUR2B.23 These cells contain K+ currents that could be activated by 100 µmol/L diazoxide or 10 µmol/L levcromakalim and inhibited by 10 µmol/L glibenclamide. We observed that application of 10 µmol/L forskolin resulted in activation of Kir6.1/SUR2B currents (Figure 2Ai). Currents increased from 163.2±15.5 pA/pF to 239.9±28.6 pA/pF (n=20; P<0.001; measured 2 minutes after forskolin application), reaching a peak response within 2 minutes, and followed by a slower and partial reduction in current. This stimulation was seen across the whole voltage range (Figure 2Aii). In contrast, Kir6.2/SUR2B currents were unaffected by forskolin in whole-cell experiments (Figure 2B). In perforated patch experiments, forskolin did increase Kir6.2/SUR2B current (data not shown), but this was not pursued further in this study. To investigate the effect of directly raising cAMP, the cell-permeant cAMP analogue 8-bromo-cAMP (2 mmol/L) was applied and was found to stimulate Kir6.1/SUR2B current. This effect was sustained until washout of the compound (Figure 2C). We confirmed that activation of Kir6.1/SUR2B was activated via PKA by using Rp-cAMPS (200 µmol/L), which competes with cAMP for binding to the regulatory subunit of PKA. When it was included in the patch pipette solution, forskolin no longer stimulated Kir6.1/SUR2B current, but there was still a slow decrease in current with time (Figure 2D). The inclusion of a phosphodiesterase inhibitor did not substantially affect the degree of Kir6.1/SUR2B current activation (Figure 2E). We explored further the nature of the slow time-dependent decrease in current in the presence of forskolin. Application of the inactive forskolin derivative 1,9'-dideoxyforskolin (10 µmol/L) did not lead to an increase in current (Figure 2E), but the inhibition of Kir6.1/SUR2B current still occurred and was reversible (data not shown). One possibility is that forskolin may block the Kir6.1/SUR2B channel pore, probably from the cytoplasmic side, in addition to its action on adenylate cyclase. This accounts for the slow inhibition observed during the application of forskolin and derivatives. Finally, we investigated the role that particulate PKA may have in channel modulation. RII-containing PKA subunits are scaffolded to the plasma membrane (and intracellular sites) through A kinase-anchoring proteins (AKAPs).27,28 This interaction can be disrupted using a peptide (Ht31). We included this peptide in the patch pipette, but it did not abrogate forskolin activation of Kir6.1/SUR2B currents (Figure 2E).
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Thus, it is apparent that Kir6.1/SUR2B currents can be regulated in a PKA-dependent fashion in the whole-cell configuration. However, such experiments do not discriminate between direct channel phosphorylation and indirect mechanisms in which PKA is an intermediary component of a signaling cascade. We used 2 assays to address this. First, we synthesized bacterial fusion proteins of domains of the channel-containing consensus sites for PKA phosphorylation. We expressed and purified the C terminus of Kir6.1 and the nucleotide binding domains of SUR2B fused to MBP. We did not examine the N terminus of Kir6.1 because it does not contain a consensus site. Figure 3A shows coomassie-stained SDS-PAGE gels loaded with the indicated proteins. We then subjected such purified protein to in vitro phosphorylation assays with PKAcat. It is clear from the autoradiograms shown in Figure 3B that MBP-Kir6.1C, MBP-SUR2B-NBD1, and MBP-SUR2B-NBD2 are substrates for PKA-mediated phosphorylation. In contrast, MBP control protein is not phosphorylated, and thus, the radioactive phosphate is likely to be incorporated into serine/threonine residues present in the fused domain. Therefore, domains of the channel complex can act as direct substrates for PKA-mediated phosphorylation. As a second assay, we performed inside-out patch experiments in which we examined single-channel NPo before and after exposure to PKAcat. In the absence of ATP and nucleotide diphosphates, there was little Kir6.1/SUR2B channel activity; however, upon application of 1 mmol/L (Mg)ATP and 0.5 mmol/L (Na)UDP, there was an immediate increase. The Kir6.1/SUR2B channel activity did not run down with time under the indicated conditions. The subsequent addition of PKAcat together with the nucleotides resulted in an additional increase in activity (Figure 4). Although it is formally possible that a membrane-bound intermediary might still exist taken together with the biochemistry, the data are highly suggestive that channel activation occurs by direct phosphorylation of the protein complex.
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Given the results shown in Figures 3 and 4
, we attempted to establish which serine and threonine residues are phosphorylated in the cytoplasmic domains of Kir6.1 and SUR2B. The consensus site recognition program in Prosite ([R/K]-[R/K]-x-[S/T] for PKA; http://ca.expasy.org/tools/scnpsite.html) was used to identify 2 PKA consensus sites (T234, S385) in Kir6.1 and 2 PKA consensus sites in SUR2B (T633, S1465). Initially, in vitro phosphorylation assays were performed on point mutations MBP-Kir6.1C-T234A, MBP-Kir6.1C-S385A, MBP-Kir6.1-T234A-S385A, MBP-SUR2B-NBD1-T633A, and MBP-SUR2B-NBD2-S1465A. All point mutations expressed comparable levels of protein compared with wild-type and could be purified in an analogous fashion. Representative autoradiograms are shown in Figure 5A, and quantitation from a number of experiments is shown in Figure 5B. Phosphorylation by PKA was abolished in MBP-Kir6.1C-T234A, MBP-Kir6.1C-S385A, MBP-Kir6.1-T234A-S385A, and MBP-SUR2B-NBD2-S1465A but not MBP-SUR2B-NBD1-T633A. The abolition of MBP-SUR2B-NBD2-S1465A is not quite significant from the quantitation, although it is clearly present from the gels. This results from the relatively lower levels of incorporation in this particular fusion compared with the others. We further investigated the ability of T633 in NBD1 to act as a substrate for PKA phosphorylation. We synthesized a shorter fusion protein (SUR2NBD1s; see Materials and Methods) and found that it was a substrate for PKA phosphorylation. The mutant T633As were not phosphorylated (Figure 5C). In addition, we synthesized short peptides (see Materials and Methods) corresponding to this region with and without the T633A mutation. The nonmutant peptide was phosphorylated to a significantly higher degree (Figure 5D). Thus, T633 does act as substrate for phosphorylation by PKA, and its phosphorylation in full-length NBD1 is probably masked by phosphorylation at other sites.
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We next analyzed the potential functional significance of these consensus residues. We generated 2 point mutations in Kir6.1 (T234A and S385A) and 2 in SUR2B (T633A and S1465A). These mutants were transiently transfected along with SUR2B (in the case of the Kir6.1 mutants) or with Kir6.1 (in the case of the SUR2B mutants) into HEK293 cells so that each channel complex contained 1 point mutation. In whole-cell recordings, all point mutations removed the stimulation in steady-state current seen with forskolin using wild-type Kir6.1 and SUR2B cells. Figure 6A shows some representative recordings, and Figure 6B shows the mean data together with statistical analysis. All the mutants expressed robust basal whole-cell currents. Furthermore, we were able to activate these mutants using a potassium channel opener (Figure 6C shows an example). The effect of inclusion of a phosphodiesterase inhibitor is shown below.
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These point mutations were further investigated by applying the PKAcat to the intracellular face of the channel during inside-out patch recordings of single-channel activity. In such studies, 3 of these point mutants had attenuated activation (S385A in Kir6.1 and T633A and S1465A in SUR2B), whereas T234A was still significantly activated (Figure 7A and 7B). However, a K-channel opener was still able to activate the currents (Figure 7A).
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5 minutes of perfusion in each condition. Dotted lines indicate closed levels and arrows indicate open levels. B, Column graph showing mean NPo/NPoc for Kir6.1/SUR2B and each of the Kir6.1 and SUR2B mutants. Data for the control are the same as in There is a potential discrepancy between the data for the T234A mutation with the different experimental configurations (ie, whole-cell with forskolin and inside-out patch recording with PKAcat). We investigated this further and used forskolin in combination with a phosphodiesterase inhibitor. Under these circumstances, there was significant activation of T234A, but S385A was not activated (Figure 8).
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Discussion |
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We have studied the regulation of the Kir6.1/SUR2B complex by PKA, and we can recapitulate many aspects of native channel regulation in a HEK293 heterologous expression system. Our results strongly suggest that regulation occurs because of direct channel phosphorylation. What kind of model then can we propose for the link between the phosphorylation of individual sites and channel activity? It is important to appreciate that a single alanine substitution actually removes 4 potential sites because the channel complex is an octomer of 4 Kir6.1 subunits and 4 SUR2B subunits. Our hypothesis is that channel activation is largely mediated via phosphorylation at S385A in Kir6.1 and T633 and S1465 residues in SUR2B. Furthermore, significantly enhanced channel activity only occurs at high levels of phosphorylation (ie, when multiple sites are phosphorylated). Such a mechanism is appealing because it would be of value in stopping channel activation from occurring because of fluctuations in basal PKA activity. In vivo, this would prevent precipitous changes in blood pressure. How well does our data support such a scheme?
We have examined the role of 4 consensus sites. Alanine substitution of S385 in Kir6.1 and S1465 in SUR2B clearly abolishes biochemical and functional evidence of regulation. T633A is convincingly implicated from the functional studies; however, the fusion protein MBP-NBD1-T633A is still significantly phosphorylated. It seems likely that other sites are phosphorylated in NBD1, and this masks phosphate incorporation at T633. Biochemical studies on a shorter fusion of this domain support this contention. It is known that PKA may potentially phosphorylate at sites such as R-X-[S/T].29 T234 in Kir6.1 seems to be of relatively less functional importance. It can be activated by PKAcat applied in inside-out patches in a quantitatively comparable fashion to wild-type Kir6.1. Furthermore, the mutant channel can be activated by forskolin in the whole-cell configuration in the presence of a phosphodiesterase inhibitor, likely to lead to a higher and more sustained rise in intracellular cAMP than forskolin alone, whereas Kir6.1 S385A is not. A further interesting biochemical feature is that phosphorylation of the Kir6.1 residues may be intrinsically cooperative as alanine substitution of either one of the residues results in a substantial reduction of signal. Thus, the functional effects arising from the T234A mutation may well occur from diminished ability to phosphorylate the S385 residue at a given level of PKA activity.
Other investigators have examined PKA modulation of the Kir6.2/SUR1 complex.30,31 The exact details of the mechanism varied between the reports and are also different from our proposed model for the Kir6.1/SUR2B complex. Gonoi et al did find evidence for a phosphorylated site on both SUR1 and Kir6.2.30 The residue identified in SUR1 was responsible for basal activity, and the residue on the channel (equivalent to our S385 residue) was responsible for activation. The residues identified by us on SUR2B are clearly involved in PKA-mediated activation, and it is not clear whether they have an effect on basal activity as well. Lin et al identified another distinct single site on the channel responsible for activity (homologous to our T234 residue).31 It is possible that mechanisms similar to those suggested by us to account for the functional effects of the T234A mutant in the Kir6.1/SUR2B complex may pertain to the Kir6.2/SUR1 complex as well. In our hands, there seems to be a fundamental difference in the mechanism of regulation of Kir6.2/SUR2B compared with Kir6.1/SUR2B.19 Also in this study, we found that activation of Kir6.2/SUR2B by forskolin only occurred in perforated patch recordings. Our data are generally comparable with that obtained in native tissues; however, 1 point on which they differ is the role of AKAPs. In native smooth muscle cells, it has been convincingly demonstrated that the Ht31 peptide abolishes forskolin and receptor-mediated activation of the native current.32 However, in HEK293 cells, it was still possible to activate the current by including PKAcat in the pipette. Thus, it is plausible that the particular cellular location of PKA may not be critical for functional activation. The observations may reflect the predominant distribution of PKA in the 2 different cell systems. It is known that the majority of endogenous PKA in HEK293 cells is present in the soluble fraction.33
Are there any precedents for multisite PKA phosphorylation in the regulation of other ion channels? One particularly well studied example is CFTR, in which a number of residues in the R domain are thought to be important for channel activation.34,35 However, activation appears to be more graded in relation to the phosphorylation status. One intriguing question is how mechanistically PKA acts to increase NPo. For example, in CFTR, phosphorylation of the R domain by PKA is essential before ATP can activate the channel by binding at the nucleotide-binding domains. The situation is different with the Kir6.1/SUR2B complex, in which channel opening is critically dependent on the provision of nucleotide diphosphates and PKA fine tunes that activity. A second mechanistic point is the potential for protein kinases to act by modulating anionic phospholipid sensitivity.36,37 It is plausible that for Kir6.2, PKA might increase the phosphatidyl inositol bisphosphate (PIP2) affinity resulting in channel activation. This is an unlikely mechanism with Kir6.1 because it already has a very high affinity for anionic phospholipids, and addition of further PIP2 does not lead to activation.19 These are interesting issues to pursue in further studies.
In summary, we have begun to unravel the molecular mechanisms of PKA regulation of Kir6.1/SUR2B. Our data implicate at least 3 sites in playing an important role in this modulation and support a model in which a large number of these sites need to be phosphorylated before substantial channel activation occurs.
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Acknowledgments |
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This work was supported by the British Heart Foundation (BHF) and the Wellcome Trust. J.P.G. is a BHF junior fellow.
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Footnotes |
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Original received October 23, 2003; revision received March 31, 2004; accepted April 2, 2004.
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References |
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