Oe K., Sperlagh B., Santha E., Matko I., Nagashima H., Foldes F. for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with KATP channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate KATP channels in patch-clamp experiments, whereas d-glucose was without effect. Overall, our STING agonist-4 data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac KATP channels.Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T. A., Coetzee, W. A. Cardiac ATP-sensitive K+ channel STING agonist-4 associates with the glycolytic enzyme complex. enterocytes is inhibited by ATP formed by the glycolytic enzyme pyruvate kinase, which was suggested to be associated with the plasma membrane (4). A structural basis for these data was provided by our finding that KATP channel subunits physically associate with some of the glycolytic enzymes (5). Using a proteomics screen and coimmunoprecipitation assays, we identified GAPDH, triosephosphate isomerase, and pyruvate kinase as KATP channel-interacting proteins. We also demonstrated that the catalytic activity of these enzymes is sufficient to regulate KATP channel activity Mouse monoclonal to LPA in the open-cell or inside-out membrane patch-clamp configuration (5). The finding that GAPDH interacts with KATP channel subunits has also been reported by others (6). In the studies described here, we demonstrate that interaction of glycolytic enzymes with KATP channel subunits is a general feature of the channel complex. Using coimmunoprecipitation assays, tandem mass spectrometry peptide analysis, and immunocytochemistry, we demonstrate association of KATP channels with enzymes throughout the glycolytic chain. We further demonstrate with patch-clamp recordings that substrates of aldolase A and pyruvate kinase led to channel inhibition in a manner that depends on their catalytic activity, whereas glucose had no effect. Our data suggest that the glycolytic pathway is an integral component of the KATP channel protein complex and that most of the glycolytic enzymes (especially those involved in the distal ATP-generating STING agonist-4 steps) are physically and functionally associated with the cardiac KATP channel. MATERIALS AND METHODS Two-hybrid protein-protein interaction screen Using bioinformatic approaches (COILS algorithm: http://www.ch.embnet.org/software/COILS_form.htm), we identified putative SUR1 and SUR2 coiled-coil (CC) domains and isolated them by PCR. Briefly, aa 947C997 of hamster SUR1 (accession number: “type”:”entrez-protein”,”attrs”:”text”:”A56248″,”term_id”:”1083191″A56248) were amplified by PCR (forward primer: 5-CCGGAATTCCGGGGAGAGGAAAGCCTCAGAGC-3; reverse primer: 5-CGCGGATCCGCGCGGGATCTTAGCTCGCTGATG-3). Similarly, the cDNA corresponding to aa 911C977 of rat SUR2 (accession number: “type”:”entrez-protein”,”attrs”:”text”:”NP_037172″,”term_id”:”19923678″NP_037172) was isolated (forward primer: 5-CGGAATTCCGCCCTCATGAATAGACAGG-3; reverse primer: 5-CGCGGATCCGCGCCAGCAGGTCTTCCAGGG-3) and subcloned (using engineered restriction sites for 10 min at 4C), the supernatant was subjected to ultracentrifugation (190,000 for 1 h at 4C), and the pellet (crude membrane fraction) was solubilized overnight at 4C in 20 mM HEPES (pH 7.4) and 0.5% Triton X-100. For coimmunoprecipitation experiments, rat hearts were rinsed in PBS (Invitrogen, Carlsbad, CA, USA) and homogenized in homogenization buffer (100 mM sucrose, 10 mM EDTA, 46 mM KCl, 5 mM NaN3, and 100 mM Tris-HCl, pH STING agonist-4 7.4, with protease inhibitor STING agonist-4 cocktail and 300 M PMSF) using a Polytron homogenizer, followed by 30 strokes in a loose-fitting Dounce homogenizer, and finally 10 strokes in a tight-fitting Dounce homogenizer. The homogenate was centrifuged (4000 for 13 min at 4C), and the supernatant was used for ultracentrifugation. Membrane fractionation was performed using Optiprep gradients (Sigma-Aldrich, St. Louis, MO, USA) as described previously (10) with modifications. In brief, the supernatant was brought to 5% Optiprep, overlaid with working solution (in mM: 250 sucrose, 1 EDTA, and 10 Tris-HCl, pH 7.4) and loaded on top of 15% Optiprep in the working solution. After centrifugation at 200,000 for 2.5 h, 7 distinct fractions were collected. Since KATP channel subunits were found in the first, third, and fourth fractions from the bottom (data not shown), these fractions were pooled and diluted with 10 mM Tris-HCl and 1 mM EDTA (pH 7.5), and membranes were recovered by centrifugation (15 min at 310,000 the identified CC domain, we subcloned the SUR2 CC domain into a bait vector and used it in a bacterial 2-hybrid screen against a rat heart cDNA library. The screen yielded several positive clones, most with unknown relevance to KATP channel function (Table 1). Interestingly, the screen identified the glycolytic enzyme GAPDH as a putative binding.