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(V)1.3, 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.