Allosterically coupled calcium and magnesium binding sites are unmasked by ryanodine receptor chimeras

Biochem Biophys Res Commun. 2008 Feb 22;366(4):988-93. doi: 10.1016/j.bbrc.2007.12.058. Epub 2007 Dec 26.

Abstract

We studied cation regulation of wild-type ryanodine receptor type 1 ((WT)RyR1), type 3 ((WT)RyR3), and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [(3)H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca(2+) titrations with (WT)RyR3 and three chimeras show biphasic activation that is allosterically coupled to an attenuated inhibition relative to (WT)RyR1. Chimeras show biphasic Mg(2+) inhibition profiles at 3 and 10 microM Ca(2+), no observable inhibition at 20 microM Ca(2+) and monophasic inhibition at 100 microM Ca(2+). Ca(2+) imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca(2+) transients with inhibition kinetics that are significantly slower than those expressing (WT)RyR1 or (WT)RyR3. Four new aspects of RyR regulation are evident: (1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, (2) Ca(2+) facilitates removal of the inherent Mg(2+) block, (3) (WT)RyR3 exhibits reduced cooperativity between H activation sites when compared to (WT)RyR1, and (4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca(2+) transients.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Regulation / drug effects
  • Allosteric Site
  • Caffeine / pharmacology
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Cells, Cultured
  • Kinetics
  • Magnesium / metabolism*
  • Magnesium / pharmacology
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / metabolism*
  • Ryanodine Receptor Calcium Release Channel / chemistry*
  • Ryanodine Receptor Calcium Release Channel / metabolism*
  • Sarcoplasmic Reticulum / drug effects
  • Sarcoplasmic Reticulum / metabolism

Substances

  • Recombinant Fusion Proteins
  • Ryanodine Receptor Calcium Release Channel
  • Caffeine
  • Magnesium
  • Calcium