The interaction of ruthenium red with surface charges controlling excitation-contraction coupling in frog sartorius.

Frog sartorii were incubated in choline Ringer solution containing different amounts of the cationic dye ruthenium red, and were subsequently superfused with ruthenium red-free solution. The contraction threshold was measured during and after the incubation at different calcium and magnesium concentrations. During incubation in ruthenium red the threshold potential is slowly shifted to more positive values depending on time of incubation and the ruthenium red concentration (10--300 microns). After ca. 40 min of incubation a saturation potential is reached. The threshold shift is already maximal (-38mV threshold potential) at 30 microns of ruthenium ret regardless of the calcium concentration up to 5 mM. Omitting calcium from the incubation solution or adding 0.5 mM magnesium instead of calcium resulted in a more negative saturation potential (-48 mV). Washing the muscle in ruthenium red-free solution for 60 min after the incubation fails to reverse the threshold shift completely. The irreversible component of the threshold shift does not depend on the divalent cation concentration during incubation as long as the saturation value during incubation is more positive than -50 mV. The contraction threshold achieved after incubation with ruthenium red is dependent on the divalent cation concentration with calcium being twice as effective as magnesium. The effect of ruthenium red is greatest at small divalent cation concentrations and not significant at 50 mM. Incubating muscles with 5 units of neuraminidase shifted the concentration threshold to more positive potentials to the same extent as incubation with ruthenium red. Subsequent treatment of the neuraminidase-treated muscles with 30 microns of ruthenium red has no further effect on contraction threshold. The alternative experiment, first incubation with ruthenium red and then treatment with neuraminidase, gives the same results. The results are explained by the interaction of ruthenium red with membrane-bound sialic acid. This interaction is thought to result in a decrease in negative charges which results in a shift of the surface potential and hence of the contraction threshold to more positive potentials.