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Incremental Ca2+ mobilization by inositol trisphosphate receptors is unlikely to be mediated by their desensitization or regulation by luminal or cytosolic Ca2+.

ABSTRACT: The kinetics of Ins(1,4,5)P3 (InsP3)-stimulated Ca2+ release from intracellular stores are unusual in that submaximal concentrations of InsP3 rapidly release only a fraction of the InsP3-sensitive Ca2+ stores. By measuring unidirectional 45Ca2+ efflux from permeabilized rat hepatocytes, we demonstrate that such quantal responses to InsP3 occur at all temperatures between 2 and 37 degrees C, but at much lower rates at the lower temperatures. Preincubation with submaximal concentrations of InsP3, which themselves evoked quantal Ca2+ release, had no effect on the sensitivity of the stores to further additions of InsP3. The final Ca2+ content of the stores was the same whether they were stimulated with two submaximal doses of InsP3 or a single addition of the sum of these doses. Such incremental responses and the persistence of quantal behaviour at 2 degrees C indicate that InsP3-evoked receptor inactivation is unlikely to be the cause of quantal Ca2+ mobilization. Reducing the Ca2+ content of the intracellular stores by up to 45% did not affect their sensitivity to InsP3, but substantially reduced the time taken for each submaximal InsP3 concentration to exert its full effect. These results suggest that neither luminal nor cytosolic Ca2+ regulation of InsP3 receptors are the determinants of quantal behaviour. Our results are not therefore consistent with incremental responses to InsP3 depending on mechanisms involving attenuation of InsP3 receptor function by cytosolic or luminal Ca2+ or by InsP3 binding itself. We conclude that incremental activation of Ca2+ release results from all-or-nothing emptying of stores with heterogeneous sensitivities to InsP3. These characteristics allow rapid graded recruitment of InsP3-sensitive Ca2+ stores as the cytosolic InsP3 concentration increases.

SUBMITTER: Beecroft MD

PROVIDER: S-EPMC1218657 | biostudies-other | 1997 Aug

REPOSITORIES: biostudies-other

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Project description: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+).

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Incremental Ca2+ mobilization by inositol trisphosphate receptors is unlikely to be mediated by their desensitization or regulation by luminal or cytosolic Ca2+.

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