Calcium-dependence of Donnan potentials in glycerinated rabbit psoas muscle in rigor, at and beyond filament overlap; a role for titin in the contractile process.

In glycerinated rabbit psoas muscle, Donnan potential measurements demonstrated that the net electric charge on the actin-myosin matrix undergoes a sharp switch-like transition at pCa(50) = 6.8. The potentials are 2 mV less negative at the lower pCa(2+) (P < 0.001). If ATP is present, the muscle contracts and breaks the microelectrode. Therefore the rigor state was studied. There is no reason to suppose a priori that a similar voltage switch does not occur during contraction, however. Calcium dependence is still apparent in muscles stretched beyond overlap (sarcomere length>3.8 µm) and is also seen in the gap filaments between the A- and I-band ends; further stretching abolishes the dependence. These experiments strongly suggest that calcium dependence is controlled initially by the titin component, and that this control is lost when titin filaments break. We suppose that that effect is mediated by the titin kinase in the M-line region and may involve the extensible PEVK region of titin. There is great interest in the electric charge on proteins in muscle within the structural system. We suggest how changes in these charges may control the calcium activation process. We also suggest some simple experimental approaches that could clarify these effects.

Ion-dependence of Z-line and M-line response to calcium in striated muscle fibres in rigor.

The calcium-dependent contraction of vertebrate skeletal muscle is thought to be primarily controlled through the interaction of the thick and thin filaments. Through measurement of the Donnan potential, we have shown that an electrical switching mechanism (sensitive to both anions and cations) is present in both A- and I-bands [1]. Here we show that this mechanism is not confined to the contractile apparatus and report for the first time the presence of M-line potentials. The Z-line responds to Ca2+ ions in a similar manner to the A-band under the same solution conditions (phosphate-chloride and imidazole buffers), even though it has no reported Ca2+ binding sites. Z-line potentials were not observed in tris-acetate buffer. The M-line has a markedly different response to any of the other subsarcomeric regions, however, and can only be detected in the phosphate-chloride buffer. Preliminary observations of the M-line potential in creatine kinase-deficient mouse muscle (phosphate-chloride buffer) reveal significant differences in the calcium-induced transitions between two of the genotypes and demonstrate definitively that it is the M-line potential that is being recorded. From these results, it seems likely that the charge response of the Z-line and M-line is being mediated by titin in an anion-dependent manner. Our evidence comes from several observations. First, the similarity between the response of the Z-line potentials to the A-band potentials, where titin is the only link between these structures and second, the differential observation of M-line and Z-line potentials in a range of buffers containing different anion(s). Both Z-line and M-line potentials were seen in phosphate-chloride buffer, but only the Z-line potentials could be detected in chloride-only (imidazole) buffer and neither was observed in the acetate buffer. The latter observations can be attributed to two sources. The first is the effect of acetate buffer on the conformation of myosin [2]; the second is the absence of binding of the M-line protein, myomesin, to titin in the absence of phosphate ions [3].

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 pCa50 6.8 in both A- and I-bands. At zero Mg2+ the pCa50 of the A-band transition is shifted to 8.0 and the I-band shows two transitions (pCa50 approximately 6.8 and approximately 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 approximately 8.5 and approximately 7.0) are evident in the A-band and three in the I-band (pCa50 approximately 8.5, approximately 7.4, approximately 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 approximately 7 and approximately 8), whilst in the I band a single transition (pCa approximately 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.