A-Kinase Anchoring Protein Targeting of Protein Kinase A and Regulation of HERG Channels
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Adrenergic stimulation of the heart initiates a signaling cascade in cardiac myocytes that increases the concentration of cAMP. Although cAMP elevation may occur over a large area of a target-organ cell, its effects are often more restricted due to local concentration of its main effector, protein kinase A (PKA), through A-kinase anchoring proteins (AKAPs). The HERG potassium channel, which produces the cardiac rapidly activating delayed rectifying K+ current (I Kr), is a target for cAMP/PKA regulation. PKA regulation of the current may play a role in the pathogenesis of hereditary and acquired abnormalities of the channel leading to cardiac arrhythmia. We examined the possible role for AKAP-mediated regulation of HERG channels. Here, we report that the PKA-RII-specific AKAP inhibitory peptide AKAP-IS perturbs the distribution of PKA-RII and diminishes the PKA-dependent phosphorylation of HERG protein. The functional consequence of AKAP-IS is a reversal of cAMP-dependent regulation of HERG channel activity. In further support of AKAP-mediated targeting of kinase to HERG, PKA activity was coprecipitated from HERG expressed in HEK cells. Velocity gradient centrifugation of solubilized porcine cardiac membrane proteins showed that several PKA-RI and PKA-RII binding proteins cosediment with ERG channels. A physical association of HERG with several specific AKAPs with known cardiac expression, however, was not demonstrable in heterologous cotransfection studies. These results suggest that one or more AKAP(s) targets PKA to HERG channels and may contribute to the acute regulation of I Kr by cAMP.
KeywordsPKA AKAP HERG Potassium channel Phosphorylation Protein interaction
Dynamic control of action potentials in cardiac tissue underlies the normal response to varying physiological demands placed on the heart. In large part, this is governed by changes in ion channel activity in response to the autonomic nervous system cholinergic and adrenergic stimuli commensurate to hemodynamic challenges. Dysregulation of this balance plays a central role in pathological conditions such as heart failure. Spatial and substrate specificity of β-adrenergic signaling is maintained by localization of protein kinase A (PKA) and phosphodiesterases to subcellular microdomains. Localization of these proteins may be achieved by scaffolding adapter proteins termed A-kinase anchoring proteins (AKAPs) (Colledge and Scott 1999; Rubin 1994; Ruehr et al. 2004). The AKAPs are a group of proteins that lack primary structure sequence homology but share function: to localize PKA to subcellular structures, substrates, and oftentimes with other members of the signaling pathway (Smith et al. 2006). The regulatory subunits (RI and RII) of PKA bind and hold the catalytic subunits (C) in an inactive state (R2C2 holoenzyme) until they bind cAMP, whereupon active catalytic portions dissociate toward target proteins (Taylor et al. 1990). The R-subunits bind to their respective RI or RII-specific AKAPs, providing local targeting of the catalytic subunit prior to its activation. The N termini of R-subunits form helix-turn-helix structures that create a binding groove for AKAPs, with specificity determined, in part, by the shallow (RII) or deep (RI) nature of the groove (Banky et al. 2003; Gold et al. 2006).
The human ether-a-go-go related gene (HERG) encodes the voltage-dependent potassium channel HERG and produces the repolarizing current I Kr (Sanguinetti et al. 1995). I Kr is important in controlling the orderly repolarization at the end of each cardiac action potential, particularly near the threshold potential of early afterdepolarizations that can trigger tachyarrhythmias. The importance of HERG/I Kr in adrenergic signaling is highlighted by the fact that disease-causing mutations of HERG often lead to a syndrome in which arrhythmias are triggered by sudden emotional or auditory stress (Schwartz et al. 2001; Wilde et al. 1999). HERG/I Kr responds to acute adrenergic signaling in a complex fashion that includes direct cAMP binding to the channel, PKA-dependent phosphorylation of HERG and phosphorylation-dependent binding to the adaptor protein 14-3-3 (Choe et al. 2006; Kagan et al. 2002; Thomas et al. 2003).
Here, we report the first evidence that acute cAMP/PKA-dependent regulation of HERG is targeted by cellular AKAP(s) to determine the degree of channel phosphorylation and biophysical response to acute cAMP stimulation. Such targeted PKA signaling to HERG may represent potential targets for future therapies in both hereditary and acquired dysregulation of the channel.
Materials and Methods
Cell Culture, Transfection and Expression Constructs
The HEK-293 cell line (from American Type Culture Collection, Manassas, VA) stably expressing C-terminal c-myc epitope-tagged HERG cDNA in pCI-Neo vector (Promega, Madison, WI) was cultured in RPMI-1640 (Mediatech, Manassas, VA) supplemented with l-glutamine, 10% fetal calf serum (HyClone, Logan, UT), and penicillin/streptomycin (Mediatech, Manassas, VA). Cultured cells were maintained in 5% CO2 humidified air at 37°C. Constructs expressing GFP fused AKAP-IS and AKAP-IS-Scr were expressed in these cells by transient transfection using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM medium (Invitrogen). AKAP-IS and AKAP-IS-Scramble constructs fused with GFP in pcDNA V5/HIS vectors were provided by Dr. John Scott (Alto et al. 2003) and PKA-R1, PKA-RII fused with 6xHIS tag in pET14b vector (Novagen, Madison, WI) were from Dr. Charles Rubin (Dong et al. 1998; Li and Rubin 1995).
Immunoprecipitation, SDS-PAGE and Immunoblot Analyses
Cell proteins were harvested for analysis as described previously (McDonald et al. 1997). Briefly, cells were lysed in ice-cold NDET buffer (150 mm NaCl, 25 mm Tris-HCl [pH 7.5], 5 mm EDTA, 1% NP-40, 0.4% deoxycholic acid, 1 mm NaF, 1 mm Na3VO4 and EDTA-free protease inhibitor cocktail tablets [Roche Pharmaceuticals, Nutley, NJ]) for 1 h with agitation. Lysates were cleared by centrifugation at 13,000 rpm for 10 min. Protein concentration was measured using the BCA method (micro-BCA, Pierce, Rockford, IL).
For immunoprecipitation, cell lysates were precleared with Ultra-Link Protein G-agarose (Pierce) for 30 min at 4°C. Supernatant was then incubated with anti-c-myc (A14G; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-HERG (C-20) antibody for 1–2 h. A nonspecific IgG was used in parallel experiments to control for nonspecific binding. Antibody–antigen complexes were precipitated by protein-G-agarose for 3 h at room temperature or overnight at 4°C. After thorough washing with PBS, proteins were eluted from the resin with 4× Laemmli sample buffer and subjected to SDS-PAGE and immunoblot analysis.
Nitrocellulose membranes were blocked with 10% fat-free milk in Tris-buffered saline (TBS) and incubated with primary antibody solution for 2 h at room temperature or overnight at 4°C. After washing with TBS-T buffer (1 × TBS 0.5% Tween-20), the membrane was incubated with horseradish peroxidase–conjugated (Pierce) or IRDye-conjugated (Rockland, Gilbertsville, PA) secondary antibody in 5% milk TBS-T buffer for 30 min. Immunoblotting results were visualized with ECL or the Odyssey infrared imaging system (Li-Cor Biotechnology, Lincoln, NE). Exposed film (Eastman Kodak, Rochester, NY) was scanned and underwent densitometric analysis. Odyssey software was used to create images and analyze the results.
PKA-Regulatory Subunit Overlay and Far-Western Blotting
PKA-RI and -RII overlay/far-Western was carried out as described by Hausken et al. (1998). Expression of PKA-RI or -RII subunit in pET14b vector (Novagen) was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3) bacteria. HIS-tagged protein was purified using Ni-NTA resin (Qiagen, Chatsworth, CA). For the overlay assay, cell samples were separated by SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% nonfat milk and 1% BSA in TBS for 30 min at room temperature. Purified PKA regulatory subunit was added to a final concentration of 0.5 μg/ml in blocking buffer, for a minimum of 3 h at room temperature. PKA-RI and -RII binding was visualized by subsequent Western blot using PKA-regulatory subunit–specific antibodies (BD Transduction Laboratories, San Diego, CA).
Immunofluorescence Analysis and Confocal Microscopy
HEK-HERG cells were plated in glass-bottomed 35-mm dishes (MatTek, Ashland, MA) at low density. Sample preparation was carried out at room temperature. Samples were rinsed with 1 x PBS three times between steps. Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 15 min and permeabilized with 0.3% Triton X-100 in PBS for 5 min. After 30-min incubation in blocking buffer (5% BSA, 0.1% Triton X-100, 1 × PBS), samples were incubated with primary antibodies diluted in blocking buffer (1:100 for PKA-RI, -RII, and c-myc antibodies from Santa Cruz Biotechnology) for 2 h. Secondary antibodies (Alexa 555 conjugated donkey anti-rabbit, Alexa 647 conjugated donkey anti-mouse antibody; Invitrogen) were reacted with samples at 1:1,000 dilution in blocking buffer for 1 h. After extensive washes, cover glasses were mounted with Gel/Mount (Biomeda, Foster City, CA). Cells were observed using the Leica (Deerfield, IL) AOBS confocal microscope at the Analytical Imaging Facility, Albert Einstein College of Medicine.
In Vitro Phosphorylation
In vitro (or back) phosphorylation was carried out as previously published (Cui et al. 2000). Briefly, HERG protein was isolated from HEK-HERG cells by immunoprecipitation. Antigen–antibody complexes were incubated with purified catalytic subunit of PKA (Sigma, St. Louis, MO) in reaction buffer containing 50 mm Tris–HCl (pH 7.0), 10 mm MgCl2, 0.1 mg/ml BSA and 10 mCi [γ-32P]-ATP for 60 min at 30°C. Proteins were washed with ice-cold NDET, separated by SDS-PAGE and transferred to nitrocellulose membrane. Relative specific activity of HERG-32P was determined by taking the ratio of the scintillation count of the 32P signal over the densitometry of the Western blot signal. To assay for coprecipitation of kinase activity with HERG, immunocomplexes were washed three times and resuspended in 110 μl of kinase reaction buffer with or without addition of 500 nm PKI (PKA inhibitor 6–22 NH2). Next, 5 μCi of [γ-32P] ATP in 10 μl of 880 μm Mg-ATP were added and incubated at 30oC for 30 min. Reactions were analyzed by SDS-PAGE, immunoblot and autoradiography.
Whole-cell patch-clamp current recordings were carried out as previously described (Kagan et al. 2002). Polished patch pipettes with tip resistances of 2–3 MΩ were used. All experiments were carried out at 20–22°C. Cells plated onto glass coverslips the night before were placed into a flow chamber mounted onto the stage of an inverted microscope equipped with epifluorescence illumination and micromanipulators to maneuver patch pipettes. Currents were measured with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA) controlled by a PC using pClamp9 software for data acquisition and analysis (Axon Instruments). Whole-cell capacitance was recorded (generally 10–30 pF) and compensated by analog circuitry of the amplifier. The calculated junction potential for our experimental solutions was 3–4 mV and was not corrected for analyses. The series resistance of 5–12 MΩ (whole cell) was compensated to 75–90% with amplifier circuitry such that the voltage errors for currents of 2 nA were <5 mV. To study I Kr, currents were elicited from a holding potential of −80 mV to depolarizing steps between –70 and +50 mV for 2 s, followed by sequential repolarizing steps to −40 mV and −120 mV. Signals were analog-filtered at 1,000 Hz and sampled at 5,000 Hz. Voltage-dependent activation data were fitted to the Boltzmann relation: I = 1/(1 + exp[(V 1/2 – V)/k]). Current densities were calculated as current (pA) divided by cell capacitance (pF). The voltage dependence of activation was obtained by plotting the normalized peak tail current against the test potential. Internal pipette solution was composed of (in mm) KCl 120, MgCl2 2, CaCl2 0.5, EGTA 5, ATP-Mg4 4, HEPES 10 (pH 7.2, osmolality 280 ± 10 mOsm/L). External solution consisted of (in mm) NaCl 150, CaCl2 1.8, KCl 4, MgCl2 1, glucose 5, HEPES 10 (pH 7.4, osmolality 320 ± 10 mOsm). Data analysis was performed with Clampfit (Axon Instruments) and Origin 7.5 (Microcal, Northampton, MA) software.
Membrane and Microsome Preparation and Velocity Gradient Centrifugation
Fresh porcine cardiac tissue (∼30 g) was homogenized using Polytron PT-2100 (Kinematica, Bohemia, NY) in ∼100 ml ice-cold lysis buffer (10 mm KCl, 2 mm MgCl2, protease inhibitors) supplemented with 0.25 m sucrose. The crude homogenate was cleared with a slow (1,000 × g) and a subsequent fast (10,000 × g) centrifugation (10 min each); the resulting supernatant was then loaded on top of a sucrose cushion (lysis buffer + 0.5 m sucrose) and centrifuged at 34,000 rpm (∼200,000 × g) for 20 min in SW 41Ti rotor (Beckman Instruments, Palo Alto, CA), yielding a membrane-rich pellet (“microsomal fraction”). Velocity gradient centrifugation was then performed using a modification of a previously described method (Okamoto et al. 2001; Sargiacomo et al. 1995). Briefly, either confluent 10-cm culture plates of HEK-HERG cells or pig cardiac microsomes (one-sixth of total material) were lysed in 0.5 ml of MBST (1% Triton X-100 + protease inhibitors in 2-(N-morpholino) ethanesulfonic acid buffered saline (MBS), pH ∼6.5). The entire lysate was loaded atop a 3.7 ml 5–40% linear sucrose gradient (in MBST) and centrifuged at 50,000 rpm (∼340,000 × g) for 16 h in an SW 60 Ti rotor (Beckman Instruments). Liquid fractions (n = 16 or 17) of equal volume were collected from the top, while the pellet was extracted with NDET buffer.
Pattern of AKAP Activity in HEK-293 Cells
AKAP-IS Peptide Affects the Distribution of PKA-RII in HEK-293 Cells
AKAP-IS Decreases PKA-Dependent Phosphorylation of HERG Channels
AKAP-IS Reverses the Acute cAMP/PKA Regulation of HERG Current
HERG Coprecipitates PKA Activity from HEK Cells
Pattern of AKAP Activity and ERG Channels from Porcine Ventricular Myocardium
Acute β-adrenergic regulation of HERG/I Kr may play an important role in the maintenance of cardiac excitability in situations of both normal and maladaptive physiology. That cAMP and PKA are involved in controlling HERG has been shown in vitro (Cui et al. 2000; Thomas et al. 1999) and indirectly from genetic studies (Choe et al. 2006; Schwartz et al. 2001; Wilde et al. 1999). There is evidence, however, that the regulatory system may be more complex in cardiac tissue and may involve additional members of a macromolecular complex (Heath and Terrar 2000; Kagan et al. 2002; Thomas et al. 2003). Here, we provide evidence that that this process may be facilitated by targeting of PKA to the vicinity of the channel by one or more PKA-RII-specific AKAPs. Inhibition of PKA-RII binding to AKAPs in HEK cells with AKAP-IS resulted in altered distribution of PKA-RII, reduced PKA-dependent phosphorylation of HERG protein and reversed PKA-dependent functional effects on the channel.
AKAPs are a structurally diverse group of proteins that bind the regulatory subunits (RII and/or RI) of PKA, thereby targeting and concentrating the enzyme to specific locales of action, generally to specific membrane domains (Colledge and Scott 1999; Rubin 1994). AKAPs often coordinate the scaffolding of various other molecules to a particular site such as protein phosphatases and phosphodiesterase in addition to the specific substrates, resulting in a macromolecular signaling complex (Appert-Collin et al. 2006; Dodge-Kafka et al. 2006; McConnachie et al. 2006). Since the first report of AKAPs (Sarkar et al. 1984), more continue to be added to the list (Feliciello et al. 2001; Ruehr et al. 2004).
PKA-RII regulatory subunit interactions with AKAPs have been the most thoroughly studied; however, RI subunits are also now recognized as targets for AKAPs (Angelo and Rubin 1998; Huang et al. 1997; Li et al. 2001). The AKAPs that have been best established in the heart include mAKAP (also known as AKAP6), AKAP15/18 (AKAP7), Yotiao (AKAP9), AKAP-Lbc (AKAP13), AKAP79 (AKAP5), gravin (AKAP12), AKAP220 (AKAP11), BIG2, MAP2B, AKAP95 (AKAP8), AKAP149/121 (AKAP1) and D-AKAP-1 (AKAP1) (Ruehr et al. 2004). For the most part, these AKAPs target PKA-RII; however, there is evidence for both PKA-RI and RII targeting in the case of D-AKAP-1 and AKAP220. In some instances, the AKAPs have been shown to enhance cAMP/PKA regulation of ion channels in the heart during periods of sympathetic stimulation such as Yotiao and KCNQ1/I Ks (Marx et al. 2002).
Our results suggest that one or more PKA-RII-specific AKAPs may be involved in mediating adrenergic and cAMP regulation of HERG. The AKAP-IS inhibitor is specific to PKA-RII subunits; therefore, we cannot rule out the potential contribution of PKA-RI-specific AKAP targeting to HERG. That ERG channels comigrated with several RI- and RII-binding proteins raises the possibility that more than one macromolecular signaling complex exists for channel regulation. If more than one AKAP could associate with HERG, adrenergic and PKA-mediated modification of channels may differentially occur at various stages of channel synthesis, assembly, trafficking and regulation of I Kr at the surface.
A pressing question that remains is which AKAP(s) is involved in targeting PKA to the HERG channel. Of the known cardiac AKAPs, we have heterologously coexpressed HERG with Yotiao, AKAP220, Ezrin, MAP2, AKAP15/18 and AKAP75/79 with no evidence of coimmunoprecipitation (supplemental data). These results may indicate that other AKAPs are involved or that some intermediary specific to cardiac myocytes is required for association. Alternatively, AKAPs may colocalize to a particular membrane environment (such as a lipid raft) that brings the channel and PKA into a close approximation without direct protein–protein interaction. In the latter case, standard coimmunoprecipitation assays would fail to detect an association even in heterologous cotransfections. That PKA is targeted via Yotiao to the KCNQ1/KCNE1 complex (Marx et al. 2002) was considered, but velocity gradient centrifugation showed no significant difference in sedimentation of RII when HERG was coexpressed with either KCNE1 or KCNE2 (supplemental data). Whether associated KCNE in vivo affects AKAP targeting remains an open question.
These results support further investigation into the nature of macromolecular signaling complexes that may regulate HERG/I Kr. Such studies should give attention to the possibility that different complexes may occur at different temporal and spatial sites of HERG biogenesis and activity. Knowledge of specific signal-transduction entities may impact future medical therapies for various cardiovascular disorders.
We thank Dr. J. D. Scott, Vollum Institute, Portland, OR for providing the AKAP-Scr, AKAP-IS and yotiao constructs and Dr. Charles Rubin, Albert Einstein College of Medicine for providing the AKAP15/18, PKA-RI and PKA-RII constructs and helpful advice on approaches to AKAP investigation. This work was funded by the National Institutes of Health (R01 HL077326 to T. V. M.) and an American Heart Association postdoctoral fellowship (to Y. L.).
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