Diethyl pyrocarbonate modification of the ryanodine receptor/Ca2+ channel from skeletal muscle.
Exposure of junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor to the histidine-specific reagent diethyl pyrocarbonate (DEPC) led to concentration- and time-dependent inactivation of ryanodine binding. The pH-dependence of the inactivation of ryanodine binding by DEPC and the reversal of this inactivation by hydroxylamine suggests the modification of histidine residue(s) by the reagent. Kinetic analysis of the time course of inactivation of ryanodine binding by DEPC suggests that the inactivation resulted from modification of a single class of histidine residue per ryanodine-binding site. The degree 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 modification. This modification decreased the total ryanodine-binding sites. DEPC modification increased the Ca(2+)-permeability of the SR vesicles. A variety of bivalent cations prevented the DEPC inactivation of ryanodine binding in a series of decreasing efficiency: Mn2+ > Ba2+ > Mg2+ > Ca2+, similar to their effectiveness in inhibiting ryanodine binding. It is suggested that a histidine residue(s) in the ryanodine receptor is involved, either in the binding of Ca2+, or in a conformational 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.
Shoshan-Barmatz
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Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
Weil
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009132:Muscles / Q000187:drug effects* / Q000378:metabolism; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012965:Sodium Chloride / Q000737:chemistry
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D012965:Sodium Chloride; D004047:Diethyl Pyrocarbonate; D002118:Calcium