Abstract

Type I, II, and III inositol-1,4,5-trisphosphate (InsP₃) receptors are expressed selectively in different cell lines and tissues. We examined whether type I, II, and III InsP₃receptors differ in ligand-binding affinity and whether such differences influence the sensitivity of Ca²⁺ stores to InsP₃. Initially, SH-SY5Y human neuroblastoma cells, AR4–2J rat pancreatoma cells, and RINm5F rat insulinoma cells were studied because these cells express predominantly (>85%) type I, II, and III receptors, respectively. Immunopurification of receptors from these cell lines and measurement of InsP₃ binding revealed that the rank order of affinity for InsP₃ was type I > type II > type III (binding sites were half-maximally saturated at 1.5, 2.5, and 22.4 nm InsP₃, respectively). Examination of Ca²⁺ store mobilization in permeabilized cells showed that InsP₃ was equipotent in SH-SY5Y and AR4–2J cells but was ∼5-fold less potent in RINm5F cells. In contrast, Ca²⁺ uptake and InsP₃-independent Ca²⁺ release were very similar in the three cell types. The binding affinity of InsP₃ in permeabilized SH-SY5Y, AR4–2J, and RINm5F cells correlated well with its potency as a Ca²⁺-mobilizing agent and with binding affinity to immunopurified type I, II, and III receptors. Thus, InsP₃ receptor binding affinity seems to influence the potency of InsP₃ as a Ca²⁺-mobilizing agent. Finally, immunopurification of type I, II, and III receptors from rat tissues revealed that the affinity differences seen in receptors purified from cultured cells are paralleled in vivo. In combination, the data from cell lines and rat tissues reveal that type I, II, and III receptors bind InsP₃ with K _d values of ∼1, ∼2, and ∼40 nm, respectively, and that the selective expression of a particular receptor type will influence the sensitivity of cellular Ca²⁺ stores to InsP₃.