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 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 K_D (nM) and B_max (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 K_D value of 1.9 nM and a B_max value of 95 fmol/mg of protein. The apparent K_D value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca²⁺ (K_D/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 K_D value similar to and a B_max value 1.8‐fold greater than those obtained for the low‐affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the K_D value was fivefold lower, whereas the B_max value was unaffected. The K_D 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.