Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca²⁺ and mediate quantal Ca²⁺ mobilization

There are three subtypes of mammalian Ins(1,4,5)P₃ (InsP₃) receptor, each of which forms an intracellular Ca²⁺ channel. Biphasic regulation of InsP₃ receptors by cytosolic Ca²⁺ is well documented in cells expressing predominantly type 1 or type 2 InsP₃ receptors and might contribute to the regenerative recruitment of Ca²⁺ release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP₃ receptors of RIN-5F cells, cells in which the InsP₃ receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca²⁺ [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP₃ receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP₃ receptors in RINm5F cells. In unidirectional ⁴⁵Ca²⁺ efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP₃ released only a fraction of the InsP₃-sensitive Ca²⁺ stores, indicating that responses to InsP₃ are quantal. Increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) from approx. 4 to 186 nM increased the sensitivity of the Ca²⁺ stores to InsP₃: the EC₅₀ decreased from 281±15 to 82±2 nM. Further increases in [Ca²⁺]_i massively decreased the sensitivity of the stores to InsP₃, by almost 10-fold when [Ca²⁺]_i was 2.4 μM, and by more than 3000-fold when it was 100 μM. The inhibition caused by 100 μM Ca²⁺ was fully reversed within 60 s of the restoration of [Ca²⁺]_i to 186 nM. The effect of submaximal InsP₃ concentrations on Ca²⁺ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca²⁺. We conclude that type 3 InsP₃ receptors of RINm5F cells mediate quantal Ca²⁺ release and they are biphasically regulated by cytosolic Ca²⁺, either because a single type 1 subunit within the tetrameric receptor confers the Ca²⁺ inhibition or because the type 3 subtype is itself directly inhibited by Ca²⁺.