title: The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum.
abstract: In this work we show that ryanodine binding to junctional sarcoplasmic reticulum (SR) membranes or purified ryanodine receptor (RyR) is inhibited in a time- and concentration-dependent fashion by prior treatment with the carboxyl reagent dicyclohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3-(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-pheny-lisoxazolium-3'-sulfonate (WRK) only slightly affects their ryanodine binding capacity. The amphipathic reagent N-ethoxy cabonyl-2-ethoxy-1,2-dihydroquinaline (EEDQ) inhibited ryanodine binding at relatively high concentrations. DCCD-modification of the SR decreased the binding affinities of the RyR for ryanodine and Ca2+ by about 3- and 18-fold, respectively. The single channel activity of SR membranes modified with DCCD and then incorporated into planar lipid bilayers is very low (5-8%) in comparison to control membranes. Application of DCCD to either the myoplasmic (cis) or luminal (trans) side of the reconstituted unmodified channels resulted in complete inhibition of their single channel activities. Similar results were obtained with the water soluble reagent WRK applied to the myoplasmic, but not to the luminal side. The DCCD-modified non-active channel is re-activated by addition of ryanodine in the presence of 250 microM Ca2+ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100 microM of Ca2+. The results suggest that a carboxyl residue(s) in the RyR is involved either in the binding of Ca2+, or in conformational changes that are produced by Ca2+ binding, and are required for the binding of ryanodine and the opening of the Ca2+ release channel.
authors.affiliation: Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
authors.forename: W
authors.initials: W
authors.lastname: Feng
authors.forename: V
authors.initials: V
authors.lastname: Shoshan-Barmatz
mesh_terms: D000818:Animals; D001665:Binding Sites; D000001:Calcimycin / Q000737:chemistry / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D002264:Carboxylic Acids / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004024:Dicyclohexylcarbodiimide / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D007555:Isoxazoles / Q000737:chemistry / Q000494:pharmacology; D008051:Lipid Bilayers; D009124:Muscle Proteins / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D002264:Carboxylic Acids; D007202:Indicators and Reagents; D007555:Isoxazoles; D008051:Lipid Bilayers; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D000001:Calcimycin; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D004024:Dicyclohexylcarbodiimide; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium