DIFFERENTIAL REGULATION OF TYPE-1 AND TYPE-3 INOSITOL TRISPHOSPHATE RECEPTORS BY CYTOSOLIC CA2+

Biphasic regulation of inositol trisphosphate (IP3)-stimulated Ca2+ mo bilization by cytosolic Ca2+ is believed to contribute to regenerative intracellular Ca2+ signals. Since cells typically express several IP3 receptor isoforms and the effects of cytosolic Ca2+ are not mediated by a single mechanism, it is important to resolve the properties of ea ch receptor subtype. Full-length rat types-1 and -3 IP3 receptors were expressed in insect Sf9 cells at levels 10-40-fold higher than the en dogenous receptors. The expressed receptors were glycosylated and asse mbled into tetramers, and binding of [H-3]IP3 to each subtype was regu lated by cytosolic Ca2+. The effects of increased [Ca2+] on native cer ebellar and type-1 receptors expressed in Sf9 cells were indistinguish able. A maximally effective increase in [Ca2+] reversibly inhibited [H -3]IP3 binding by approx. 50 % by decreasing the number of IP3-binding sites (B-max) without affecting their affinity for IP3. The effects o f Ca2+ on type-3 receptors were more complex: increasing [Ca2+] first stimulated [H-3]IP3 binding by increasing B-max, and then inhibited it by causing a substantial decrease in the affinity of the receptor for IP3. The different effects of Ca2+ on the receptor subtypes were not a consequence of limitations in the availability of accessory proteins or of artifactual effects of Ca2+ on membrane structure. We conclude that Ca2+ can inhibit IP3 binding to types-1 and -3 IP3 receptors alth ough by different mechanisms, and that IP3 binding to type-3 receptors is stimulated at intermediate [Ca2+]. A consequence of these differen ces is that, at resting cytosolic [Ca2+], type-3 receptors are more se nsitive than type-1 receptors to IP3, but the situation reverses at hi gher cytosolic [Ca2+]. Such differences may be important in generating the spatially and temporally complex changes in cytosolic [Ca2+] evok ed by receptors Linked to IP3 formation.