Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain

R. A. Padua, J. I. Nagy, J. D. Geiger

Research output: Contribution to journalArticle

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Abstract

Abstract: [3H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCI) and low (200 mM KCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca2+, Mg2+, β,γ‐methylene‐adenosine 5′‐triphosphate (AMP‐PCP), and caffeine. In 1.0 M KCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent KD (nM) and Bmax (fmol/mg of protein) values of 2.4 and 34, respectively, for the high‐affinity site and 19.9 and 157, respectively, for the low‐affinity site. Unlabeled titration/equilibrium measurements gave a single high‐affinity site with a KD value of 1.9 nM and a Bmax value of 95 fmol/mg of protein. The apparent KD value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca2+ (KD/Ca2+= 14 nM), inhibited by Mg2+ (IC60= 5.0 mM), and unaffected by AMP‐PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with a KD value similar to and a Bmax value 1.8‐fold greater than those obtained for the low‐affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the KD value was fivefold lower, whereas the Bmax value was unaffected. The KD value derived from association and dissociation analysis was 2.4‐fold greater in 200 mM KCI compared with 1.0 M KCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg2+ inhibited binding was increased by 3.8‐fold, whereas the affinity of the activation site for Ca2+ was reduced by 13‐fold. Addition of caffeine in the presence of low salt increased the affinity of Ca2+ activation by 1.7‐fold. The inhibitory effect of Mg2+ on [3H]‐ryanodine binding in the presence of 200 mM KCI was reversed by AMP‐PCP and caffeine with apparent EC50 values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.

Original languageEnglish (US)
Pages (from-to)2340-2348
Number of pages9
JournalJournal of Neurochemistry
Volume62
Issue number6
DOIs
StatePublished - Jun 1994

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Keywords

  • Calcium channel
  • Ionic strength
  • Membrane bindina
  • Ryanodine receptor

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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