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

ABSTRACT: There are three subtypes of mammalian Ins(1,4,5)P(3) (InsP(3)) receptor, each of which forms an intracellular Ca(2+) channel. Biphasic regulation of InsP(3) receptors by cytosolic Ca(2+) is well documented in cells expressing predominantly type 1 or type 2 InsP(3) receptors and might contribute to the regenerative recruitment of Ca(2+) release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP(3) receptors of RIN-5F cells, cells in which the InsP(3) receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca(2+) [Hagar, Burgstahler, Nathanson and Ehrlich (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 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%) InsP(3) receptors in RINm5F cells. In unidirectional (45)Ca(2+) efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP(3) released only a fraction of the InsP(3)-sensitive Ca(2+) stores, indicating that responses to InsP(3) are quantal. Increasing the cytosolic free [Ca(2+)] ([Ca(2+)](i)) from approx. 4 to 186 nM increased the sensitivity of the Ca(2+) stores to InsP(3): the EC(50) decreased from 281+/-15 to 82+/-2 nM. Further increases in [Ca(2+)](i) massively decreased the sensitivity of the stores to InsP(3), by almost 10-fold when [Ca(2+)](i) was 2.4 microM, and by more than 3000-fold when it was 100 microM. The inhibition caused by 100 microM Ca(2+) was fully reversed within 60 s of the restoration of [Ca(2+)](i) to 186 nM. The effect of submaximal InsP(3) concentrations on Ca(2+) mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca(2+). We conclude that type 3 InsP(3) receptors of RINm5F cells mediate quantal Ca(2+) release and they are biphasically regulated by cytosolic Ca(2+), either because a single type 1 subunit within the tetrameric receptor confers the Ca(2+) inhibition or because the type 3 subtype is itself directly inhibited by Ca(2+).

SUBMITTER: Swatton JE

PROVIDER: S-EPMC1220613 | biostudies-other | 1999 Nov

REPOSITORIES: biostudies-other

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Project description:Submaximal concentrations of inositol 1,4,5-trisphosphate (InsP3) rapidly release only a fraction of the InsP3-sensitive intracellular Ca2+ stores, despite the ability of further increases in InsP3 concentration to evoke further Ca2+ release. The mechanisms underlying such quantal Ca2+ mobilization are not understood, but have been proposed to involve regulatory effects of cytosolic Ca2+ on InsP3 receptors. By examining complete concentration-effect relationships for InsP3-stimulated 45Ca2+ efflux from the intracellular stores of permeabilized hepatocytes, we demonstrate that, at 37 degrees C, responses to InsP3 are quantal in Ca(2+)-free media heavily buffered with either EGTA or BAPTA [1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid]. Lower concentrations of InsP3 were used to examine the kinetics of Ca2+ mobilization at 2 degrees C, because at the lower temperature the stores were more sensitive to InsP3: the concentration of InsP3 causing half-maximal Ca2+ release (EC50) after a 30 s incubation decreased from 281 +/- 37 nM at 37 degrees C to 68 +/- 3 nM at 2 degrees C. At 2 degrees C, the EC50 for InsP3-stimulated Ca2+ mobilization decreased as the duration of exposure to InsP3 was increased: the EC50 was 68 +/- 3 nM after 30 s, and 29 +/- 2 nM after 420 s. InsP3-stimulated Ca2+ mobilization is therefore non-quantal at 2 degrees C: InsP3 concentration determines the rate, but not the extent, of Ca2+ release. By initiating quantal responses to InsP3 at 37 degrees C and then simultaneously diluting and chilling cells to 2 degrees C, we demonstrated that the changes that underlie quantal responses do not rapidly reverse at 2 degrees C. At both 37 degrees C and 2 degrees C, modest increases in cytosolic Ca2+ increased the sensitivity of the stores to InsP3, whereas further increases were inhibitory; both Ca2+ effects persisted after prior removal of ATP. We conclude that the effects of Ca2+ on InsP3 receptors are unlikely either to be enzyme-mediated or to underlie the quantal pattern of Ca2+ release evoked by InsP3.

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

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