CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a Sendai virus vector.

Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3- -rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (H2-DIDS), but not by a specific CFTR inhibitor, CFTR(inh)-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.

M-LDH serves as a regulatory subunit of the cytosolic substrate-channelling complex in vivo.

Nucleoside diphosphate kinase A (NDPK-A) regulates the alpha1 isoform of the AMP-activated protein kinase (AMPK alpha1) selectively, independent of [AMP] and surrounding [ATP], by a process termed substrate channelling. Here, we show, using a range of empirically validated biochemical techniques, that the muscle form (M-LDH or LDH-A) and the heart form (H-LDH or LDH-B) of lactate dehydrogenase are physically associated with the liver cytosolic substrate-channelling complex such that M-LDH associates with NDPK-A, AMPK alpha1 and casein kinase 2 (CK2), whereas H-LDH associates with local NDPK-B. We find that the species of LDH bound to the substrate-channelling complex regulates the in vivo enzymatic activities of both AMPK and CK2, and has a downstream effect on the phospho-status of acetyl CoA carboxylase, a key regulator of cellular fat metabolism known to be a part of the cytosolic substrate-channelling complex in vivo. We hypothesise that the regulatory presence of LDH in the complex couples the substrate-channelling mechanism to both the glycolytic and redox states of the cell, allowing for efficient sensing of cell metabolic status, interfacing with the substrate-channelling complex and regulating the enzymatic activities of AMPK and CK2, two critical protein kinases.

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site.

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.

Protein kinase CK2 acts as a signal molecule switching between the NDPK-A/AMPK alpha1 complex and NDPK-B.

Previously we elucidated the molecular interaction between the nucleoside diphosphate kinase A (NDPK-A)/AMP-activated protein kinase (AMPK) alpha1 complex, discovering a process we termed "substrate channeling." Here, we investigate the protein-protein interaction of the substrate channeling complex with the pleiotropic protein kinase, CK2 (formerly casein kinase 2). We show that CK2 is part of the NDPK-A/AMPK alpha1 complex under basal (background AMPK activity) conditions, binding directly to each of the complex components independently. We report that when S122 on NDPK-A is phosphorylated by AMPK alpha1 in vivo, (i.e., stimulation of AMPK using either metformin or phenformin) initiating the substrate channeling mechanism, the catalytic subunit of CK2 (CK2alpha) is expelled from the complex and translocates to bind NDPK-B, a closely related but independent isoform of NDPK. Thus, we find that the AMPK-dependent phospho-status of S122 on NDPK-A determines whether CK2alpha swaps partners between NDPK-A and NDPK-B. This is the first reported linkage between NDPK-A and NDPK-B via a phosphorylation pathway and could explain the complex biology of NDPK. This study also offers an explanation as to how CK2alpha exclusion mutations (S120A or S122D of NDPK-A) on NDPK-A might have implications in cancer biology and general cellular energy metabolism.

Nucleoside diphosphate kinase A as a controller of AMP-kinase in airway epithelia.

This review integrates recent understanding of a novel role for NDPK-A in two related directions: Firstly, its role in an airway epithelial cell when bound to the luminal (apical) membrane and secondly in the cytosol of many different cells (epithelial and non-epithelial) where an isoform-specific interaction occurs with a regulatory partner, AMPKalpha1. Thus NDPK-A is present in both a membrane and cytosolic environment but in the apical membrane, its roles are not understood in detail; preliminary data suggest that it co-localises with the cystic fibrosis protein (CFTR). In cytosol, we find that NDPK-A is coupled to the catalytic alpha1 isoform of the AMP-activated protein kinase (AMPKalpha subunit), which is part of a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. We find that ATP is located within this complex and 'fed' from NDPK to AMPK without ever 'seeing' bulk solution. Importantly, the reverse can also happen such that AMPK activity can be made to decline when NDPK-A 'steals' ATP from AMPK. Thus we propose a novel paradigm in NDPK-A function by suggesting that AMP-kinase can be regulated by NDPK-A, independently of AMP.

Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha 1.

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16 to 20 kDa) including two well-characterized isoforms (NDPK-A and -B). NDPK catalyzes the conversion of nucleoside diphosphates to nucleoside triphosphates, regulates a diverse array of cellular events, and can act as a protein histidine kinase. AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that responds to the cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of acetyl coenzyme A carboxylase 1 (ACC1), a regulator of cellular fatty acid synthesis. We recently reported that NDPK-A (but not NDPK-B) selectively regulates the alpha1 isoform of AMPK independently of the AMP concentration such that the manipulation of NDPK-A nucleotide trans-phosphorylation activity to generate ATP enhanced the activity of AMPK. This regulation occurred irrespective of the surrounding ATP concentration, suggesting that "substrate channeling" was occurring with the shielding of NDPK-generated ATP from the surrounding medium. We speculated that AMPK alpha1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK alpha1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK alpha1 and that the phosphorylation status of S122, but not S144, determines whether substrate channeling can occur. We report the cellular effects of the S122 mutation on ACC1 phosphorylation and demonstrate that the presence of E124 (absent in NDPK-B) is necessary and sufficient to permit both AMPK alpha1 binding and substrate channeling.

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes.

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.

Glyceraldehyde 3-phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal K(ATP) channel.

Cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal K(ATP)-channel protein complex. A polypeptide of approximately 42 kDa was immunoprecipitated with an anti-SUR2A antibody from guinea-pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti-Kir6.2, anti-SUR2A and anti-GAPDH antibodies showed that GAPDH is a part of the sarcolemmal K(ATP)-channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two-hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch-clamp electrophysiology showed that GAPDH regulates K(ATP)-channel activity irrespective of high intracellular ATP, by producing 1,3-bisphosphoglycerate, a K(ATP)-channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal K(ATP)-channel protein complex, where it couples glycolysis with the K(ATP)-channel activity.

A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase alpha1 in liver and lung.

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16-20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK alpha1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the alpha1 (but not alpha2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK alpha1-mediated phosphorylation is present in mammalian cells.

Hypoxia-induced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal KATP channels.

The opening of sarcolemmal and mitochondrial ATP-sensitive K(+) (KATP) channels in the heart is believed to mediate ischemic preconditioning, a phenomenon whereby brief periods of ischemia/reperfusion protect the heart against myocardial infarction. Here, we have applied digital epifluorescent microscopy, immunoprecipitation and Western blotting, perforated patch clamp electrophysiology, and immunofluorescence/laser confocal microscopy to examine the involvement of KATP channels in cardioprotection afforded by preconditioning. We have shown that adult, stimulated-to-beat, guinea-pig cardiomyocytes survived in sustained hypoxia for approximately 17 min. An episode of 5-min-long hypoxia/5-min-long reoxygenation before sustained hypoxia dramatically increased the duration of cellular survival. Experiments with different antagonists of KATP channels, applied at different times during the experimental protocol, suggested that the opening of sarcolemmal KATP channels at the beginning of sustained hypoxia mediate preconditioning. This conclusion was supported by perforated patch clamp experiments that revealed activation of sarcolemmal KATP channels by preconditioning. Immunoprecipitation and Western blotting as well as immunofluorescence and laser confocal microscopy showed that the preconditioning is associated with the increase in KATP channel proteins in sarcolemma. Inhibition of trafficking of KATP channel subunits prevented preconditioning without affecting sensitivity of cardiomyocytes to hypoxia in the absence of preconditioning. We conclude that the preconditioning is mediated by the activation and trafficking of sarcolemmal KATP channels.

ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimulated insulin secretion.

As part of our research into the liver-directed gene therapy of Type I diabetes, we have engineered a human hepatoma cell line (HEPG2ins/g cells) to store and secrete insulin to a glucose stimulus. The aim of the present study was to determine whether HEPG2ins/g cells respond to glucose via signaling pathways that depend on ATP-sensitive potassium channels (KATP). Using patch-clamp electrophysiology with symmetrical KCl solutions, the single-channel conductance of KATP was 61pS. KATP was inhibited by ATP (1 mM) or cAMP (50 microM) applied to the cytosolic side of the membrane. Single KATP channels and macroscopic whole-cell currents were inhibited by glucose (20 mM) and glibenclamide (20 microM) and were activated by diazoxide (150 microM). Immunoprecipitation and Western blot analysis confirmed the presence of Kir6.2 KATP channel subunit protein in HEPG2ins/g and HEPG2ins cells. Using radioimmunoassay techniques, we report that exposure of the cells to tolbutamide (100 microM) resulted in an increase in insulin secretion from 0.3 +/- 0.05 to 1.8 +/- 0.2 pmol insulin/10(6) cells and glibenclamide (20 microM) from 0.4 +/- 0.06 to 2.1 +/- 0.3 (n=4), similar to what is seen on glucose (20 mM) stimulation. Diazoxide (150 microM) completely inhibited glucose-stimulated insulin release. Glucose 20 mM and glibenclamide 100 microM increased intracellular Ca2+ level in the HEPG2ins/g cells. However, glucose 20 mM did not stimulate a rise in intracellular Ca2+ in the un-transfected parent cell-line HEPG2. We used confocal microscopy to confirm that glucose (20 mM) stimulated the release of insulin from the fluorescently labeled secretion granules in the cells. Furthermore, glibenclamide (20 microM) also stimulated the release of insulin from fluorescently labeled secretion granules, and diazoxide (150 microM) blocked that stimulated release of insulin. Our results suggest that HEPG2ins/g cells respond to glucose via signaling pathways that depend on KATP, similar to a normal pancreatic beta cell.

Chronic mild hypoxia protects heart-derived H9c2 cells against acute hypoxia/reoxygenation by regulating expression of the SUR2A subunit of the ATP-sensitive K+ channel.

Chronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO2) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca2+ loading in heart-derived H9c2 cells. The number of ATP-sensitive K+ (K(ATP)) channels were increased in cells exposed to PO2 = 100 mm Hg relative to cells exposed to PO2 = 144 mm Hg. This was due to an increase in transcription of SUR2A, a K(ATP) channel regulatory subunit, but not Kir6.2, a K(ATP) channel pore-forming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1alpha and dominant negative HIF-1beta suggested that the HIF-1-signaling pathway did not participate in observed PO2-mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO2 = 100 mm Hg but not the effect of PO2 = 20 mm Hg. LY 294002 and PD 184 352 prevented PO2-mediated regulation of K(ATP) channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO2 = 100 mm Hg, and a decrease of PO2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1alpha-independent signaling cascade leading to an increase in SUR2A protein, a higher density of K(ATP) channels, and a cellular phenotype more resistant to acute metabolic stress.

Large conductance Ca2+-activated K+ channels sense acute changes in oxygen tension in alveolar epithelial cells.

The rise in alveolar oxygen tension (PO(2)) that occurs as the newborn infant takes its first breaths induces removal of liquid from the lung lumen due to ion transport across the alveolar epithelium and the activity of alveolar Na(+) channel (ENaC). In the present study, we have aimed to identify an ion conductance in alveolar epithelial A549 cells that responds to acute changes in PO(2). Variation in PO(2) did not affect single-channel ENaC activity. However, in these cells we have detected single-channel conductance having properties similar to those of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Reverse transcriptase-polymerase chain reaction and Western blotting demonstrated presence of alpha-BKCa channel subunit and iberiotoxin, a blocker of BK(Ca) channels, inhibited whole cell K(+) current. Chronic changes in PO(2) did not affect expression, recruitment, or function of BK(Ca) channels in A549 cells. In contrast, acute changes of PO(2) regulated the BK(Ca) channel activity by controlling the channel mean open time. This effect of PO(2) was insensitive to inhibitor of flavoproteins, diphenylene iodinium. In addition, decrease in PO(2) and iberiotoxin induced membrane depolarization and Ca(2+) oscillations in A549 cells. We conclude that BK(Ca) channels serve as oxygen sensors in human alveolar A549 epithelial cells.

M-LDH serves as a sarcolemmal K(ATP) channel subunit essential for cell protection against ischemia.

ATP-sensitive K(+) (K(ATP)) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K(+) channel core, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of channel gating. Here, we have shown that the muscle form (M-LDH), but not heart form (H-LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal K(ATP) channel by interacting with the Kir6.2 subunit via its N-terminus and with the SUR2A subunit via its C-terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M-LDH in the channel protein complex was required for opening of K(ATP) channels during ischemia and ischemia-resistant cellular phenotype. We conclude that M-LDH is an integral part of the sarcolemmal K(ATP) channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the K(ATP) channels activity that is essential for cell protection against ischemia.

17Beta-estradiol regulates expression of K(ATP) channels in heart-derived H9c2 cells.

OBJECTIVES: The main objective of the present study was to establish whether 17beta-estradiol (E2) regulates expression of cardiac adenosine triphosphate-sensitive potassium (K(ATP)) channel. BACKGROUND: Based on our previous studies that demonstrate gender-specific differences in sarcolemmal K(ATP) channels, we have hypothesized that the main estrogen, E2, may regulate expression of cardiac K(ATP) channels. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Kir6.2 and sulfonylurea receptor 2A (SUR2A) subunits was performed on total ribonucleic acid (RNA) from rat embryonic heart-derived H9c2 cells. Immunoprecipitation and Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was done on membrane fraction of H9c2 cells. Whole cell electrophysiology and digital epifluorescent Ca(2+) imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2. RESULTS: The RT-PCR revealed higher levels of SUR2A, but not Kir6.2, messenger RNA (mRNA) in E2-treated, relative to untreated, cells. Increase of the level of only the SUR2A subunit could change the number of sarcolemmal K(ATP) channels only if the Kir6.2 is in excess over SUR2A. Indeed, RT-PCR analysis demonstrated considerably lower levels of SUR2A mRNA compared with Kir6.2 mRNA. Significantly higher levels of both Kir6.2 and SUR2A protein subunits were found in the membrane fraction of E2-treated cells compared with untreated ones, and the density of current evoked by pinacidil (100 microM), a K(ATP) channel opener, was significantly higher in E2-treated compared with untreated cells. To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca(2+) in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca(2+) loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 microM), but not by 5-hydroxydecanoate (50 microM). CONCLUSIONS: In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates K(ATP) channel formation and protects cardiac cells from hypoxiareoxygenation.

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner.

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.

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

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.