Potassium ions promote hexokinase-II dependent glycolysis

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K⁺. The K⁺ dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K⁺ by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K⁺ re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of K_V1.3, a voltage-gated K⁺ channel, lowered cellular K⁺ content and the glycolytic activity of HEK293 cells. Our findings unveil K⁺ as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K⁺ channels and cell metabolism.