Calcium dependence of Donnan potentials in rigor:: the effects of [Mg2+] and anions in isolated rabbit psoas muscle fibres

Contraction in vertebrate striated muscle is known to be dependent upon the binding of calcium ions to the regulatory protein troponin C (TnC). Our electrical (Donnan potential) studies of the subsarcomeric regions have revealed an electrical switching mechanism, which is sensitive to both cation concentration and to particular anions. In a buffer containing phosphate and chloride ions and at 2.7 mM Mg2+ we observe a single charge transition at pCa(50) 6.8 in both A-and I-bands. At zero Mg2+ the pCa(50) of the A-band transition is shifted to 8.0 and the I-band shows two transitions (pCa(50) similar to 6.8 and similar to 8.2). Increasing [Mg2+] to 4.5 mM produces a complex effect between pCas 7 and 9 in both bands. All effects are abolished at 9 mM Mg2+. In a chloride-only buffer (imidazole) at zero Mg2+ the direction of the charge transitions is reversed. In addition, two transitions (pCa(50) similar to 8.5 and similar to 7.0) are evident in the A-band and three in the I-band (pCa(50) -8.5, -7.4, -6.7). in the presence of Mg2+, again the effects of pCa upon the Donnan potential are complex. In the A-band at 2.7 mM Mg2+ two transitions of opposite sign predominate (pCa similar to 7 and similar to 8), whilst in the I band a single transition (pCa similar to 8.3) occurs in the same direction as that observed in phosphate buffer. At 4.5 mM Mg2+ the 'W' shape observed in the corresponding phosphate buffer is preserved in both bands with similar pCa(60)s. This shape is also apparent in the 9 mM Mg2+ solution. In these two buffer systems, the magnitude of the charge change in terms of electron binding is far larger than expected from simple Ca2+/Mg2+ binding to troponin. In an acetate-only buffer, however, the Donnan potentials of the A-band and I-band were very similar in magnitude and the charge change across the full pCa curve is close to the expected value for Ca2+/Mg2+ binding to troponin We speculate that titin has a role in the calcium activation of striated muscle in vertebrates for four reasons. First, the effects of long-term storage of the glycerinated muscle; second, the action of [Mg2+] ions; third the effect of anions; and fourth, our published and unpublished observations of sarcomere-length dependence. We also demonstrate the validity of our methodology, relating the charge transitions that we observe to cation-binding studies of a more traditional nature.