The action of carboxyl modifying reagents on the ryanodine receptor/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum

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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 dicy-clohexylcarbodiimide (DCCD). Exposure of the membrane-bound RyR to the water soluble carboxyl reagents 1-ethyl-3 (3(dimethylamino) propyl carbodiimide (EDC) or N-ethyl-phenylisoxazolium-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 μM Ca2+ and is stabilized in a sub-conductance state. With caffeine, ryanodine re-activated the channel in the presence of 100 μM 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.

Original languageEnglish
Pages (from-to)85-93
Number of pages9
JournalMolecular Membrane Biology
Volume13
Issue number2
DOIs
StatePublished - 1 Jan 1996

Keywords

  • Ca release channel
  • Carboxyl modifying reagents
  • Ryanodine receptor
  • Sarcoplasmic reticulum

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