Comparison of the effects of fluoride on the calcium pumps of cardiac and fast skeletal muscle sarcoplasmic reticulum: evidence for tissue-specific qualitative difference in calcium-induced pump conformation

Abstract

Comparison of the effects of fluoride (NaF, 1–10 mM) on the catalytic and ion transport functions of the Ca 2+-ATPase in sarcoplasmic reticulum (SR) vesicles isolated from rabbit cardiac and fast-twitch skeletal muscles revealed similarities as well as striking tissue-specific differences depending on the experimental conditions employed. Short preincubation (3 min at 37°C) of cardiac or fast muscle SR with fluoride in the absence of Ca 2+ and ATP prior to initiating enzyme turnover by simultaneous addition of Ca 2+ and ATP to the assay medium resulted in a strong inhibitory effect of fluoride on ATP-energized (oxalate-facilitated) Ca 2+ uptake and Ca 2+-ATPase activity. On the other hand, when turnover was initiated by the addition of ATP to SR preincubated with fluoride in the presence of Ca 2+ but in the absence of ATP, fluoride caused concentration-dependent stimulation of active Ca 2+ uptake by fast muscle SR with no appreciable change in Ca 2+-dependent phosphoenzyme (EP) formation (from ATP) or Ca 2+-ATPase activity but inhibition of active Ca 2+ uptake by cardiac SR with concomitant inhibition of EP formation and Ca 2+-ATPase activity. Exposure of cardiac or fast muscle SR to fluoride in the presence of both Ca 2+ and ATP resulted in concentration-dependent stimulatory effect of fluoride on Ca 2+ uptake with no change in EP formation or Ca 2+-ATPase activity; this effect diminished substantially at saturating oxalate concentration in the assay. Assessment of the effects of deferoxamine (1 mM) and exogenous aluminum (10 μM) did not indicate a requirement for aluminum in the inhibitory or stimulatory effect of fluoride. These results suggest that (a) the Ca 2+ and ATP-deprived (E 1/E 2) but not the Ca 2+ plus ATP-liganded (CaE 1ATP) conformation of the SR Ca 2+-ATPase is susceptible to inhibition by fluoride in both cardiac and fast muscle; (b) the Ca 2+-bound conformation (CaE 1) of the SR Ca 2+-ATPase is susceptible to inhibition in cardiac muscle but is refractory to fluoride in fast muscle; and (c) the stimulatory effect of fluoride is largely secondary to its ability to mimic the action of oxalate in intravesicular Ca 2+ trapping when the fluoride-resistant enzyme is turning over normally. Fluoride inhibited phosphorylation of the Ca 2+-free enzyme by P i in cardiac and fast muscle SR indicating that fluoride sensitivity of the phosphorylation site of the SR Ca 2+-ATPase is similar in cardiac and fast muscle. In cardiac SR, disruption of the functional interaction between Ca 2+-ATPase and its regulatory protein phospholamban, through phosphorylation of the latter (by cAMP kinase) did not alter the fluoride sensitivity of the Ca 2+-bound enzyme (CaE 1). These results, coupled with the refractoriness of CaE 1ATP to fluoride in cardiac and fast muscle SR, suggest that a tissue-specific difference in the accessibility (reactivity) of the nucleotide binding site to fluoride upon Ca 2+ binding to the enzyme may account for the observed difference in fluoride sensitivity of the cardiac versus fast muscle enzyme - i.e., when the ATPase is in CaE 1 conformation, its ATP binding site is ‘fluoride-reactive’ in the cardiac enzyme but is ‘fluoride-resistant’ in the fast muscle enzyme.