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 pCa506.8 in both A-and I-bands. At zero Mg2+the pCa50of the A-band transition is shifted to 8.0 and the I-band shows two transitions (pCa50~6.8 and ~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 (pCa50~8.5 and ~7.0) are evident in the A-band and three in the I-band (pCa50~8.5, ~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 ~7 and ~8), whilst in the I band a single transition (pCa ~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 pCa50s. 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the glycerinated muscle; second, the action of [Mg2+] ions; third the effect of anions; and fourth, our published and unpublished observations of sarcomere-length dependence.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.