The cardiac KATP channel associates with the glycolytic enzyme complex

Cardiac ATP‐sensitive potassium (K ATP ) channels are primarily gated by high‐energy nucleotides, a property that allows them exquisite sensitivity to changes in intracellular energy metabolism. An emerging view is that the K ATP channel is a multi‐subunit protein complex, and that the associated proteins provide an additional layer of regulation. Using bioinformatic approaches, we identified putative coiled‐coil (CC) domains in SUR subunits. We utilized these CC‐domains as baits in a two‐hybrid screen against a rat cardiac cDNA library and identified two glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and the SUR CC‐domain was confirmed using GST pull‐down assays. We further demonstrated the interaction with full‐length SUR subunits with co‐immunoprecipitation assays using heterologous expression assays. Mass spectrometry was performed with K ATP channel subunit immunoprecipitates obtained from rat membrane fractions, which identified glycolysis as the most enriched biological process. Using co‐immunoprecipitation approaches with rat cardiac membrane preparations, we confirmed interaction for several glycolytic enzymes (aldolase, enolase, fructose 6 phosphate kinase and hexokinase). These glycolytic enzymes were co‐localized with K ATP channel subunits in isolated cardiac myocytes, as determined with immunocytochemistry. The catalytic activity of aldolase and pyruvate kinase inhibited K ATP channel activity in inside‐out patch clamp experiments, whereas D‐glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP generating steps) with cardiac K ATP channels.