Specific perforation of muscle cell membranes with preserved SR functions by saponin treatment

The effects of saponin on Xenopus and frog skeletal muscle fibres were examined. The twitch of Xenopus single fibres was first potentiated slightly and then irreversibly abolished by 5-10 microgram/ml of saponin. Treatment with saponin at 5-10 microgram/ml or higher concentrations for 30 min resulted in perforation of the muscle cell membrane, indicated by the following evidence. (i) Fibres became responsive to contractile activating solutions with a pCa-tension relationship similar to that of mechanically skinned fibres. (ii) Removal and re-introduction of MgATP became effective in bringing fibres into rigor and the relaxed state, respectively. (iii) After the saponin treatment large contractions due to Ca release from the SR could be elicited by substitution of Cl for methanesulphonate in the medium. (iv) The treatment decreased the optical path length across the fibre, indicating loss of soluble proteins. (v) The lattice spacing of myofilaments was wider after the treatment as in mechanically skinned fibres. Contractile response of mechanically skinned fibres and their SR responses such as Ca uptake, Ca-induced Ca release and Cl-induced Ca release were not affected by treatment with 50 microgram/ml saponin for 30 min, while 150 microgram/ml or higher concentrations severely impaired by the SR functions. It is possible, therefore, to make chemically skinned skeletal muscle fibres in which the functions of the SR are preserved by applying 10-50 microgram/ml saponin.