Russell M. Crawford

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 ~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.

Gender-specific difference in cardiac ATP-sensitive K+channels ☆

OBJECTIVES The main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K(+) (K(ATP)) channels. BACKGROUND Recently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as K(ATP) channels. METHODS A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca(2+) imaging were performed on isolated guinea pig ventricular cardiomyocytes. RESULTS The RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 microM), a K(ATP) channel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single K(ATP) channel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca(2+) loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in K(ATP) channel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required. CONCLUSIONS This study has demonstrated that female tissue expresses higher levels of functional cardiac K(ATP) channels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.

17β-Estradiol regulates expression of KATPchannels in heart-derived H9c2 cells ☆

Abstract Objectives The main objective of the present study was to establish whether 17β-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 μM), 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 μM), but not by 5-hydroxydecanoate (50 μM). 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.

M-LDH serves as a sarcolemmal KATP channel subunit essential for cell protection against ischemia

ATP‐sensitive K+ (KATP) 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 KATP 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 KATP channels during ischemia and ischemia‐resistant cellular phenotype. We conclude that M‐LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the KATP channels activity that is essential for cell protection against ischemia.

Subaerial exposure and changes in the stability of intertidal estuarine sediments

Abstract The surface cohesive strength of intertidal sediments at six sites on two estuaries throughout the period of a single diurnal subaerial exposure was investigated. Stations at different tidal heights were examined at each site. Hourly measurements were made using a cohesive strength meter which provides an ordinal measure of sediment stability. The sediment was most easily eroded immediately after the emersion of the mudflat and there was little difference between the initial stability of the mud at different sites or stations. As the subaerial exposure continued the sediment of mid-shore and high-shore stations began to increase in stability. No significant change was found at low-shore stations before the return of the tide. The surface density of diatom populations was also intermittently measured at each site. The greatest increase in sediment stability occurred at high-shore stations and this was most extreme where there were also dense populations of epipelic diatoms present at the surface of the sediment. This supports laboratory evidence that the extracellular products of diatom populations of sufficient density increase the stability of intertidal sediments.

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 ~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.

Glyceraldehyde 3‐phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal KATP channel

Cardiac sarcolemmal ATP‐sensitive K+ (KATP) 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 KATP‐channel protein complex. A polypeptide of ∼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 KATP‐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 KATP‐channel activity irrespective of high intracellular ATP, by producing 1,3‐bisphosphoglycerate, a KATP‐channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal KATP‐channel protein complex, where it couples glycolysis with the KATP‐channel activity.

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.

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, AMPKα1. 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 (AMPKα 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.

CFTR, chloride concentration and cell volume: could mammalian protein histidine phosphorylation play a latent role?

A considerable body of evidence indicates that the intracellular chloride concentration ([Cl−]i) is an important regulatory signal in epithelial ion transport. [Cl−]i regulates the open channel probability of sodium and chloride channels, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis, the activity of sodium‐coupled chloride entry pathways and G‐protein activity. Cell volume goes awry in epithelial cells bearing mutant forms of the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR); however, the pathways that mediate this [Cl−]i effect at the apical membrane of polarized epithelia are unknown. Recently, we proposed a mechanism for the transduction of in vitro chloride concentration into a phosphorylation signal to proteins within the apical membrane of respiratory epithelia. Our studies show that an apically enriched plasma membrane fraction from a variety of species, including sheep, human and mouse airway, contains at least two membrane‐bound protein kinases which exhibit a number of novel properties. Firstly, the phosphate is located on histidine residues within different families of proteins; one kinase(s) utilizes GTP rather than ATP as a phosphate donor and each kinase has its own unique profile of membrane protein phosphorylation (which itself varies with anion species). Secondly, both kinases mediate Cl−‐dependent phosphorylation of an apical membrane protein around the established physiological values for [Cl−]i in airway epithelial cells (∼40 mm); associated phosphatases also alter the net phosphoprotein profile of the apical membrane. These findings are reviewed and their potential roles explored in relation to the pathogenesis of CF using the control of cell volume as a model for disrupted cellular function in CF‐affected epithelia.