Endogenous MLC2 phosphorylation and Ca²⁺-activated force in mechanically skinned skeletal muscle fibres of the rat

Abstract

A method has been developed for measuring the level of phosphorylation of myosin regulatory light chains (MLC2) by the endogenous myosin light chain kinase in mechanically skinned skeletal muscle fibres. The method was used to characterize the endogeous MLC2 phosphorylation capacity of single fast-twitch fibres from the rat and to investigate the relationship between the endogenous MLC2 phosphorylation and the Ca²⁺-activated force. The results show that (1) about 50% of MLC2 were ³²P-phosphorylated after activation of the skinned fibre preparation by 30 μM [Ca²⁺] for longer than 30 s, but that there was variability between fibres; (2) most of the endogenous phosphorylating system diffused out of the skinned fibre preparation after 5 min exposure to an aqueous solution; (3) the MLC2 phosphorylation by the endogenous phosphorylating system followed with a delay of the order of 1–2 s after the sudden rise in [Ca²⁺] from below 10 nM to 30 μM; and (4) the sensitivity of the contractile apparatus to Ca²⁺ was markedly increased when the MLC2 were phosphorylated by the endogenous phosphorylating system following a rise in [Ca²⁺]. The K _d for MgATP of the endogenous MLC2 phosphorylating system was estimated to be less than 300 μM. These results unequivocally demonstrate that prolonged activation of the fast-twitch muscle fibre leads to increased Ca²⁺ sensitivity of the contractile apparatus and that mechanically skinned fibres can be successfully used to study the regulation of the endogenous MLC2 phosphorylation capacity at single muscle fibre level.