Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote

Channel activity of the calcium release channel from skeletal muscle, ryano dine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity wa s measured after incorporating channels into planar lipid bilayers. Optimal ly and suboptimally calcium-activated calcium release channels were inactiv ated by the application of protamine to the cytoplasmic side of the channel . Recovery of channel activity was not observed while protamine was present . The addition of protamine bound to agarose beads did not change channel a ctivity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentrati on of the medium. The block of channel activity by protamine could be rever sed either by removal by perfusion with buffer or by the addition of hepari n to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C2C12 mouse muscle cells prevented caffeine-induced intracel lular calcium release. The results suggest that protamine acts on the ryano dine receptor in a similar but opposite manner from heparin and that protam ine can be used as a potent, reversible inhibitor of ryanodine receptor act ivity.