Creatine kinase is physically associated with the cardiac ATP-sensitive K+ channel in vivo

Abstract

Cardiac sarcolemmal ATP-sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside-out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea-pig cardiac membrane fraction with the anti-SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at approximately 48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti-SUR2A immunoprecipitate and the cross reactivity between an anti-CK antibody and the anti-SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.

Figure 1. Creatine kinase regulate K_ATP channels activity

A, B) Recording of K_ATP channel activity in membrane patch treated with ADP (1 mM), ADP (1 mM) plus phosphocreatine (3 mM), and again with ADP (1 mM) (A) or treated with ATP (1 mM), ATP (1 mM) plus creatine (3 mM) and again with ATP (1 mM) (B). Holding potential: -60 mV. Doted lines correspond to the zero current levels, A1 and B1. Channel activity expressed as NPo under conditions in (A) and (B). Vertical bars represent mean ± se (n=4 for each). *P<0.01. C) Membrane currents evoked by identical families of 400 ms voltage pulses in cells filled with pipette solution containing either ATP (1 mM) or ATP (1 mM) plus creatine (3 mM) or ADP (1 mM) plus phosphocreatine (3 mM). Arrow points to the zero current level. C1. Current-voltage relationships corresponding to recording on (C), C2. Current densities at 80 mV for conditions in (C). Vertical bars represent mean ± se (n = 4-5). *P<0.01.

Figure 2. Anti-SUR2A antibody and immunoprecipitation

A) Western blotting analysis of imunoprecipitate pellets from untransfected (UT), transfected and cotransfected A549 cells. Note that apart from antibody fragments, SUR2A signal was detected only in cotransfected cells; that is, cells expressing SUR2A subunit. B) Coomasie blue stain of immunoprecipitate pellets obtained from cardiac membrane fraction precipitated with 40 μg anti-SUR2A antibody. Note the presence of proteins migrating at ∼38 kDa, ∼48 kDa, and ∼150 kDa. C) Western blot of anti-SUR2A immunoprecipitate pellets from guinea pig cardiac membrane fraction with the anti-SUR2A and anti-Kir6.2 (raised against the synthetic peptide consisting of residues 33 to 47, ARFVSKKGNCNVAHK, in the Kir6.2 protein) antibodies. Note that single signals in both cases and no cross-reactivity with any other proteins within 35-190 kDa range. HC-heavy chain.

Figure 3. CK is present in anti-SUR2A immunoprecipitate

A) Creatine kinase assay with commercial CK (Sigma). B) Creatine kinase assay with anti-SUR2A immunoprecipitate of cardiac membrane fraction. Note that CK activity is recovered in pellets in an antibody concentration-dependent manner. C) Graph representing MALDI-TOF mass spectrum of tryptic mass fingerprint obtained from ∼48 kDa migrating protein. The protein was identified as muscle form of creatine kinase.

Figure 4. CK is associated with cardiac K_ATP channel protein complex

A) Coomasie blue stain of commercial muscular CK (Sigma) running at 48 kDA (b) and corresponding Western blot (w) with anti-SUR2A antibody. Note a lack of cross-reactivity between the anti-SUR2A antibody and CK. B) Western blotting of anti-SUR2A immunoprecipitate with anti-CK (anti-SUR2A IP) and anti-CK immunoprecipitate with anti-SUR2A (anti-CK IP) antibody of guinea pig cardiac membrane fraction. Note single “right” size signals (48 kDa for CK and 150 kDa for SUR2A) in both cases. HC = heavy chain.

Figure 5. CK directly associates with the SUR2A subunit

A) Creatine kinase assay with anti-SUR2A or anti-Kir6.2 immunoprecipitate of untransfected A549 cells (UT) or cells transfected with Kir6.2 plus CK (Kir6.2), SUR2A plus CK (SUR2A), and SUR2A/Kir6.2 plus CK (SUR2A/Kir6.2). Anti-Kir6.2 antibody was used for immunoprecipitation of Kir6.2 cells, whereas for all other groups anti-SUR2A antibody was applied. Note that CK activity is present only in SUR2A and SUR2A/Kir6.2 group of cells. B) Western blotting of anti-Kir6.2 and anti-SUR2A immunoprecipitate of A549 cells (same cell groups and methods as in (A). Note that single “right” size (48 kDa) band was observed only in SUR2A and SUR2A/Kir6.2 group of cells.