Functional coupling between glycolysis and sarcoplasmic reticulum Ca²⁺ transport

To investigate whether the energy derived from glycolysis is functionally coupled to Ca²⁺ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting ⁴⁵Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular ⁴⁵Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca²⁺-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated ⁴⁵Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca²⁺ pump. Addition of a soluble hexokinase ATP trap eliminated ⁴⁵Ca transport fueled by exogenous ATP but had markedly less effect on ⁴⁵Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more ⁴⁵Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca²⁺ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca²⁺ homeostasis by driving the SR Ca²⁺ pump.