Ionic strength dependence of calcium, adenine nucleotide, magnesium, and caffeine actions on ryanodine receptors in rat brain.

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 KCl) and low (200 mM KCl) 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+, beta, gamma-methylene-adenosine 5'-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCl 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+ (IC50 = 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCl 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 KCl 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 KCl compared with 1.0 M KCl buffer conditions.(ABSTRACT TRUNCATED AT 250 WORDS)