Excitation-contraction coupling in frog sartorius and the role of the surface charge due to the carboxyl group of sialic acid.

Frog sartorius muscle fibres were incubated with the enzyme neuraminidase which is known to remove surface-bound sialic acids. The sialic acid content of the incubation media was analysed, and the relationship between the threshold of contraction and the altered pH and divalent cation concentration was investigated. The threshold potential of fibres treated with 3.3, 5 or 6.7 units of neuraminidase (at pH 5.5 and 30 degrees C for 2 h) was more positive than that of the control muscle fibres incubated under the same conditions, but without the enzyme. The potential shift is positively correlated with the enzyme concentration and with the amount of sialic acid released. After incubation with 5 units of neuraminidase the potential shift rose to +8.5 mV, depending on [Ca2+]0, [Mg2+]0 and pH. The threshold shift is greatest at low divalent cation concentration (0.5 mM), and not significant at high concentrations of divalent cations (50 mM). In both neuraminidase-treated and control muscles, the effectiveness of Mg2+ is half of that of Ca2+. The dependence of the contraction threshold on pH in the range 5.5--10 is even more pronounced in enzyme-treated than in control muscle fibres. Resting potential, time-course and overshoot of action potential are not affected by treatment with neuraminidase. Threshold shifts are explained by shifts of an external surface potential upon variation of [Ca2+]0, [Mg2+]0 and pH. Treating the muscles with neuraminidase diminishes the net negative charge density, and hence shifts the surface potential to more positive values, by release of negatively charged sialic acid. The different effectiveness of Ca2+ and Mg2+ is ascribed to their different effectiveness of Ca2+ Mg2+ is ascribed to their different binding behaviour towards the negative surface charges.