Abstract
An investigation of the effects of a number of inositol trisphosphate analogues on the transient kinetics of Ca2+ release from cerebellar microsomes was undertaken. All the analogues investigated could release the total Ca2+ content of the inositol 1, 4,5-trisphosphate (Ins(1,4,5)P3) mobilizable Ca2+ store; however, their potencies were substantially reduced compared to Ins(1,4,5)P3. The concentration required to induce half-maximal Ca2+ mobilization was 0.14 microM for Ins(1,4,5)P3, 1.8 microM for 3-deoxyinositol 1,4, 5-trisphosphate (3-deoxyInsP3), 1.0 microM for 2,3-dideoxyinositol 1, 4,5-trisphosphate (2,3-dideoxyInsP3), 24 microM for 2,3, 6-trideoxyinositol 1,4,5-trisphopshate (2,3,6-trideoxyInsP3), and 2.9 microM for inositol 2,4,5-trisphosphate (Ins(2,4,5)P3). In all cases and for all concentrations tested, the inositol trisphosphate analogues induced biphasic transient release of Ca2+, which could fit to a biexponential equation assuming two independent processes. The rate constants calculated for the release process were much larger for Ins(1,4,5)P3 than the other inositol trisphosphates (the fast phase rate constant varying from 0.3 to 1.6 s-1 and the slow phase from 0.01-0.5 s-1, at concentrations between 0.03 and 20 microM Ins(1,4,5)P3). The rate constants for all other inositol trisphosphates did not appear to exceed 0.4 s-1 for the fast phase and 0.1 s-1 for the slow phase at their highest concentrations tested. The maximum amplitudes for Ca2+ release by the two phases appeared to be similar for all inositol trisphosphates (approximately 45% for the fast phase and approximately 55% for the slow phase). On comparing the rate constants for Ca2+ release at inositol trisphosphate concentrations for the analogues which all induced the same extent of Ca2+ release, it was apparent that the rates of release were independent of the extent of Ca2+ release. As the extent of Ca2+ release can be related to degree of occupancy of the binding sites, it is evident that different analogues which occupy the binding site of the receptor to the same extent can induce Ca2+ to be released at different rates. We explain this conclusion in terms of partial agonism where inositol phosphates can induce two (or more) occupied states of the channel.
Highlights
(the InsP3 receptor) causing the elevation of cytosolic Ca2ϩ concentrations (1)
In this study we have investigated the effects of a variety of inositol trisphosphate analogues on the transient kinetics of Ca2ϩ release with the view to understanding how these ligands influence the mechanism of channel opening
In the hope of gaining further insights into how this channel operates, we employed stopped-flow spectrofluorimetry to study the transient kinetics of Ca2ϩ release from cerebellar microsomes using a number of different InsP3 analogues
Summary
(the InsP3 receptor) causing the elevation of cytosolic Ca2ϩ concentrations (1). This activates a diverse multitude of cellular processes which depend on the cell type (1). A more elaborate approach to investigating InsP3-induced Ca2ϩ release is to study the transient kinetics of this process Such studies using permeabilized hepatocytes, basophilic leukemia cells, cerebellar microsomes, and purified-reconstituted InsP3 receptors have shown InsP3-induced Ca2ϩ release to be a relatively fast and biphasic process (9 –12). These studies have proved useful in aiding our understanding into the mechanism of channel opening. The use of InsP3 analogues in studies of InsP3-induced Ca2ϩ release and binding to the channel have helped shed light on our understanding of the pharmacological and functional properties of this transport protein (3, 13) Many of these analogues are able to release Ca2ϩ to the same extent and in a similar manner to Ins(1,4,5)P3, albeit with much lower affinities/potencies and have been classified as “full agonists” (3). In this study we have investigated the effects of a variety of inositol trisphosphate analogues (all of which are full agonists) on the transient kinetics of Ca2ϩ release with the view to understanding how these ligands influence the mechanism of channel opening
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