DIETHYL PYROCARBONATE MODIFICATION OF THE RYANODINE RECEPTOR CA2-MUSCLE( CHANNEL FROM SKELETAL)

Exposure of junctional sarcoplasmic reticulum (SR) membranes or purifi ed ryanodine receptor to the histidine-specific reagent diethyl pyroca rbonate (DEPC) led to concentration- and time-dependent inactivation o f ryanodine binding. The pH-dependence of the inactivation of ryanodin e binding by DEPC and the reversal of this inactivation by hydroxylami ne suggests the modification of histidine residue(s) by the reagent. K inetic analysis of the time course of inactivation of ryanodine bindin g by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The d egree of inactivation of ryanodine binding by DEPC was decreased when high NaCl concentrations were present in the modification medium. The binding affinities for ryanodine and Ca2+ were not altered by DEPC mod ification. This modification decreased the total ryanodine-binding sit es. DEPC modification increased the Ca2+-permeability of the SR vesicl es. A variety of bivalent cations prevented the DEPC inactivation of r yanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > M g2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine bin ding. It is suggested that a histidine residue(s) in the ryanodine rec eptor is involved, either in the binding of Ca2+ or in a conformationa l change that may be required for Ca2+ binding to its binding site(s). This modification of the ryanodine receptor resulted in inactivation of ryanodine binding and activation of Ca2+ release.