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

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Abstract
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.