Subcellular localization of ryanodine receptors in rat brain

Abstract

Subcellular fractions of rat brain were used to determine the subcellular localization of ryanodine receptors. [ 3H]Ryanodine binding in purified cortical, cerebellar and hippocampal synaptosomes was up to 3.6-fold higher compared with mitochondrial fractions. The density of sites ( B max) in hippocampal mossy fiber synaptosomes (249 fmol/mg protein) was 3.7-fold greater than in cortical synaptosomes (67 fmol/mg protein) and binding affinity was approximately 2-fold greater in the former ( K D, 6.1 nM) than the latter ( K D, 3.1 nM). At single sub-saturating concentrations of [ 3H]ryanodine, binding was 1.6-fold higher in mossy fibers compared with total hippocampal synaptosomes. [ 3H]Ryanodine binding sites were distributed similarly in subfractions of cortical synaptosomes and microsomes from discontinuous sucrose density gradients. An enrichment of sites was found in the lightest fractions containing the lowest activities of plasma membrane (5′-nucleotidase) and endoplasmic reticulum (glucose 6-phosphatase) enzyme markers when data for microsomal and synaptosomal subfractions were expressed as activity/binding per mg protein and when data for synaptosomal subfractions were expressed as a percentage of total activity/binding in collected fractions. Thus, ryanodine receptors appear to be concentrated in presynaptic terminals where they may play a major role in neurotransmitter release, and appear to be localized either in a specialized endoplasmic reticulum subcompartment or a distinct subcellular organelle.