Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary

Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10⁻¹¹ m Ca²⁺ and 2.7×10⁻⁹ m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10⁻⁵ m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10⁻⁴ m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10⁻⁴ m Ca²⁺ buffered with 10mm EGTA. At 1.3×10⁻⁷ m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10⁻⁴ m.