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)P-3 (InsP(3)) receptor, eac h of which forms an intracellular Ca2+ channel. Biphasic regulation of InsP (3) receptors by cytosolic Ca2+ is well documented in cells expressing pred ominantly type 1 or type 2 InsP(3) receptors and might contribute to the re generative recruitment of Ca2+ release events and to limiting their duratio n in intact cells. The properties of type 3 receptors are less clear. Bilay er recording from InsP(3) receptors of RIN-5F cells, cells in which the Ins P(3) receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca2+ [Hagar, Burgstahler, Nathanson and Ehr lich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP(3) receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera fru giperda (Sf9) cells expressing only single receptor subtypes to calibrate t he immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP(3) receptors in RINm5F cells. In unidirection al Ca-45(2+) efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP(3) released only a fraction of the InsP(3)-sensitiv e Ca2+ stores, indicating that responses to InsP(3) are quantal. Increasing the cytosolic free [Ca2+] ([Ca2+](i)) from approx. 4 to 186 nM increased t he sensitivity of the Ca2+ stores to InsP(3): the EC50 decreased from 281 /- 15 to 82 +/- 2 nM. Further increases in [Ca2+](i) massively decreased th e sensitivity of the stores to InsP(3), by almost 10-fold when [Ca2+](i) wa s 2.4 mu M, and by more than 3000-fold when it was 100 mu M. The inhibition caused by 100 mu M Ca2+ was fully reversed within 60 s of the restoration of [Ca2+](i) to 186 nM. The effect of submaximal InsP(3) concentrations on Ca2+ mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca2+. We conclude that type 3 InsP(3) receptors of RINm5F cells mediate quantal Ca2+ release and they are biphasically regulat ed by cytosolic Ca2+, either because a single type 1 subunit within the tet rameric receptor confers the Ca2+ inhibition or because the type 3 subtype is itself directly inhibited by Ca2+.