Heterogeneity of microsomal Ca²⁺ stores in chicken purkinje neurons

Chicken cerebellum microsomes were subfractionated on isopycnic, linear sucrose (15-50%) density gradients. The distribution of four markers of intracellular, rapidly-exchanging Ca²⁺ stores, i.e. the Ca²⁺ pump, the receptors for inositol 1,4,5-trisphosphate (IP₃) and ryanodine (Ry), and calsequestrin (CS, an intralumenal, high capacity Ca²⁺ binding protein) was investigated biochemically and immunologically. In the cerebellum, high levels of these markers are expressed by one of the cell types, the Purkinje neuron. Heavy subfractions were enriched in both CS and Ry receptor, intermediate subfractions in the IP₃ receptor, while the Ca²⁺ pump was present in both inter mediate and heavy subfractions. Intact cells and pelleted subfractions were examined by conventional and immuno-electron microscopy (immuno-gold labeling of ultrathin cryosections with anti-CS and anti-IP₃ receptor antibodies). Of the strongly CS-labeled, moderately dense-cored vacuoles (calciosomes) recently described in chicken Purkinje neurons only partly exhibited labeling for the IP₃ receptor as well, and the rest appeared negative. The latter were enriched in a heavy subfraction of the gradient where Ry receptors were also concentrated, whereas the CS-rich vacuoles in an intermediate subfraction were almost always IP₃ receptor-positive. The population of CS-rich calciosomes of chicken Purkinje neurons appears therefore to be molecularly heterogeneous, with a part responsive to IP₃ and the rest possibly sensitive to Ry.