Functional Coupling Between Glycolysis and Sarcoplasmic Reticulum Ca ²⁺ Transport

Abstract

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 Ca(2+)-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.

Excitation-contraction coupling and mitochondrial energetics

“…Furthermore, ATP generation from SR-associated glycolytic enzymes supports SR Ca 2+ -ATPase activity [219]. The SR Ca 2+ -ATPase, in fact, seems to be the most sensitive ATPase in response to a drop in the free energy released from ATP hydrolysis (ΔG ∼p ), which may directly affect cardiac contractility [105,130,196,197,199].…”

The Muscle-specific Calmodulin-dependent Protein Kinase Assembles with the Glycolytic Enzyme Complex at the Sarcoplasmic Reticulum and Modulates the Activity of Glyceraldehyde-3-phosphate Dehydrogenase in a Ca2+/Calmodulin-dependent Manner

“…Thus, these studies point to a previously unrecognized role for the multifunctional CaMKII in regulating membrane ATP through targeting and activation of glycolytic enzymes such as GAPDH in response to the calcium signal. Although, PGK and GAPDH normally exist in a complex and at the SR membrane (20,39,(41)(42)(43), we were unable to detect this in our assay conditions, because this association is fragile and disrupted in 0.1 M KCl (44).…”

“…GAPDH was found to be distributed in muscle cytosol and the SR membrane, and ultrastructural localization studies have also revealed that GAPDH along with other glycolytic enzymes are bound to the cytoplasmic face of the SR membrane in skeletal muscle (33,38). A role for GAPDH together with 3-phosphoglycerate kinase (PGK) has been previously established in the production of ATP from GAP, NAD ϩ , P i , and ADP at the level of the SR membrane (19,20,39,40). In addition, a recent study demonstrated that synaptic vesicles were capable of generating local ATP via the membrane-associated GAPDH system, which could support the accumulation of the excitatory neurotransmitter glutamate even during significantly reduced global cellular ATP concentrations (41).…”

“…Glycolytic intermediate GAP is converted to 1,3-BPG by GAPDH-PGK complex, and this reaction is accompanied by substrate level phosphorylation of ADP to form ATP and NADH (40). ATP and/or NADH regulates RyR and Ca-ATPase (17,20,21,45,46 …”

Neuronal nitric oxide synthase negatively regulates xanthine oxidoreductase inhibition of cardiac excitation-contraction coupling

“…We prepared SR fractions by using modifications of the methods of Xu et al (12). Whole hearts from C57BL6 mice (WT, n ϭ 6) were pooled and homogenized three times (15 s each time) in a polytron in three volumes of ice-cold 1ϫ lysis buffer (Cell Signaling Technology, Beverly, MA; catalog no.…”