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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 29, 2001 as doi:10.1096/fj.01-0466fje. |
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Tayside Institute of Child Health, Ninewells Hospital and Medical School, University of Dundee, and
* Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, Scotland, UK
2Correspondence: Tayside Institute of Child Health, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK. E-mail: a.jovanovic{at}dundee.ac.uk
SPECIFIC AIMS
Cardiac ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cell with membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the cardiac KATP channel protein complex. Therefore, we used a coimmunoprecipitation strategy on native and recombinant cardiac KATP channels, Western blotting, MALDI-TOF analysis, and patch clamp electrophysiology, to test this hypothesis.
PRINCIPAL FINDINGS
1. CK regulates cardiac KATP channel activity
Upon excision of a membrane patch from guinea pig ventricular cardiomyocyte in an ATP-free environment, vigorous openings of KATP channels were observed (not shown). Addition of phosphocreatine (3 mM) in the presence of the endogenous channel opener ADP (1 mM) on the intracellular face of the excised membrane patch closed the channels, whereas creatine (3 mM) in the presence of the endogenous channel blocker ATP (1 mM) opened them. We also measured membrane currents from a guinea pig ventricular myocyte in a whole-cell configuration. When ATP (1 mM) alone was present in the pipette solution, the steady-state voltage-current (I-V) relationship was in an N shape due to the strong inward rectification of Ik1 channels and absence of active KATP channels. However, when creatine (3 mM) plus ATP (1 mM) was added into the pipette solution, outward K+ current was significantly increased at potentials more positive than -70 mV, and the inward rectification of the I-V relationship became much weaker. In contrast, when phosphocreatine (3 mM) plus ADP (1 mM) were present in the pipette solution, whole-cell membrane currents were similar to those obtained when the pipette solution contained ATP alone.
2. Coimmunoprecipitation of cardiac membrane fraction shows that KATP channel complex is associated with the polypeptide of CK size
To immunoprecipitate proteins associated with the cardiac KATP channels, we used anti-SUR2A antibody raised against a SUR2A epitope (residues 321–334). Coomassie blue staining of the anti-SUR2A immunoprecipitate of cardiac membrane fraction revealed polypeptides with sizes corresponding to Kir6.2, SUR2A, and CK (
48 kDa). The appearance of these polypeptide bands was blocked by incubation with the corresponding antigenic peptide, and these proteins did not precipitate if the membrane fraction was probed with a non-KATP channel antibody. Western blotting analysis using the anti-SUR2A antibody and the anti-Kir6.2 antibody (raised against the residues 33 to 47 in the Kir6.2 protein) identified p38 kDa and p150 pkDa polypeptides as Kir6.2 and SUR2A.
3. Evidence that CK is associated with the cardiac KATP channel
The CK activity was present in anti-SUR2A immunoprecipitation pellets, but no activity was observed when the experimental protocol was applied without the antibody. The level of CK activity in the precipitate correlated well with the concentration of antibody. The mass spectrum of tryptic mass fingerprints of p48 kDa was identified as muscle form creatine kinase. On the other hand, no cross-reactivity between the anti-SUR2A antibody and purified CK was observed (Fig. 1
A). Western blotting of cardiac membrane anti-SUR2A immunoprecipitate with an anti-CK antibody revealed a single signal at the expected size of CK (Fig. 1B
). Conversely, the Western blotting of cardiac membrane anti-CK immunoprecipitate with an anti-SUR2A antibody revealed a single signal at the expected size of SUR2A (Fig. 1B
).
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4. SUR2A subunit associates with CK
A549 cells were cotransfected with genes encoding Kir6.2 and/or SUR2A plus the muscle form of CK cloned from mouse heart. CK assay revealed CK activity only in anti-SUR2A immunoprecipitate from cells transfected either with SUR2A plus CK or SUR2A/Kir6.2 plus CK, but not in immunoprecipitate from untransfected cells or cells transfected with Kir6.2 plus CK (Fig. 2
A). Similar results were obtained using Western blotting analysis of the immunoprecipitates with the CK antibody. A single band of 48 kDa was obtained only in immunoprecipitate from cells cotransfected with SUR2A/CK and SUR2A/Kir6.2/CK, but not in immunoprecipitate from cells without gene encoding the SUR2A subunit (Fig. 2B
).
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CONCLUSIONS
In the present study, we have tested the possibility that CK activity may change the probability of KATP channels opening. Inside-out configuration of the patch clamp technique allowed direct application of CK substrates on the KATP channel protein complex and excluded the possibility that the response of the channel might be due to the action of a cytosolic/unbound fraction of CK. CK catalyzes phosphocreatine + ADP <——> creatine + ATP reaction, and this enzyme thereby has the potential to both close and open KATP channels. Our findings that CK substrates activate or inhibit KATP channels opening agree with the hypothesis that CK is within close proximity of the channels or bound to the sarcolemma.
To examine whether CK is a part of the cardiac KATP channel complex in cardiac myocytes, we applied a coimmunoprecipitation strategy with the antibody specific for the SUR2A subunit. If CK is tightly associated with the SUR2A subunit, then it is expected to coprecipitate with the antibody. Coomassie blue staining of the anti-SUR2A immunoprecipitate revealed polypeptides with sizes corresponding to Kir6.2, SUR2A, and CK (48 kDa). First, we have demonstrated the presence of CK activity in anti-SUR2A immunoprecipitation pellets. Second, Western blotting of cardiac membrane anti-SUR2A immunoprecipitate with an anti-CK antibody revealed a single polypeptide migrating at the CK size. Third, SUR2A was identified in anti-CK immunoprecipitate of cardiac membrane fraction. Fourth, the obtained mass spectrum of tryptic mass fingerprints of p48 kDa was identified as muscle form creatine kinase (CK). Taken together, therefore, these results strongly indicate that 48 kDa protein is actually CK physically associated with the cardiac KATP channel protein complex.
To define the exact subunit of the KATP channel that interacts directly with CK, we have applied an immunoprecipitation strategy at the level of recombinant proteins. Both CK assay and Western blotting with the anti-CK antibody revealed presence of CK only in cells (co)transfected with SUR2A subunit, suggesting that CK physically associates specifically with this subunit.
Recently, a unique model of cardiac KATP channel regulation involving adenylate kinase (AK) and CK as major components of the system was proposed. It has been suggested that AK-mediated opening of the KATP channels (AMP plus ATP-induced opening of KATP channels) is reversed by CK activity (ADP plus phosphocreatine-induced closing of KATP channels). The present result that CK is a part of the KATP channel protein complex supports this model. Physical association between AK and CK with KATP channels actually means that the main intracellular producers of ATP (CK) and ADP (AK) form a complex with the KATP channel that transduces ATP and ADP levels into the changes in membrane excitability. Under physiological conditions, CK catalyzes the production of ATP from phosphocreatine and ADP, maintaining a high ATP/ADP ratio around the channel microenvironment and keeping the channel in a closed state despite the presence of AK. During severe metabolic stress in the heart, CK velocity dramatically decreases together with a decrease in the phosphocreatine/ATP ratio. These processes both promote a drop in production of ATP with a concomitant increase in ADP levels within channel vicinity, catalyzed by AK or even by CK itself, leading to opening of cardiac KATP channels. Thus, the complex of AK and CK with cardiac KATP channels would allow highly regulated transduction of metabolic status into the membrane excitability. However, to fully understand AK- and CK-mediated regulation of cardiac KATP channels, further investigations addressing protein–protein interaction between AK/CK and Kir6.2/SUR2A are required as well as functional studies to elucidate the physiological significance of the AK/CK-Kir6.2/SUR2A protein complex.
In conclusion, we have demonstrated that CK is an integral part of the cardiac KATP channel protein complex in vivo where it may regulate the activity of KATP channels (Fig. 3
). The data obtained provide a new avenue for investigating relationships between cardiac metabolism and cardiac membrane excitability.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0466fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0466fje 
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