Inositol 1,3,4,5-tetrakisphosphate binding sites in neuronal and non-neuronal tissues. Properties, comparisons and potential physiological significance

Abstract

1. Ins(1,3,4,5)P4 binding sites were studied in cerebellar and hepatic microsomes from rat, and in bovine adrenal-cortical microsomes. 2. At pH 7.0, all three tissues showed specific binding, with Ins(1,3,4,5)P4 being the most potent competing ligand of those tested [which included Ins(1,4,5)P3, Ins(1,3,4,5,6)P5 and InsP6] and Scatchard analysis suggested two sites; a site with high affinity and high specificity [Kd (1-6) x 10(-9) M] and a site with low affinity and low specificity [Kd (2-6) x 10(-7) M]. 3. At pH 5.5, cerebellar and bovine adrenal microsomes showed similar binding properties: two affinities with a similar specificity for Ins(1,3,4,5)P4 as at pH 7.0. 4. However, when assayed in a low-ionic strength acetate-based buffer at pH 5.0, cerebellar microsomes retain specific Ins(1,3,4,5)P4 binding sites, whereas bovine adrenal and hepatic microsomal binding sites lose much of their specificity, as InsP6 and Ins(1,3,4,5,6)P5 are equally as potent as Ins(1,3,4,5)P4. 5. Pi (25 mM), which is frequently included in Ins(1,3,4,5)P4 binding assays, had a small inhibitory effect on binding of cerebellar and adrenal microsomes at pH 5.5, but a large effect at pH 7.0, so that a considerable decrease occurs in the amount of specific binding at pH 5.5 compared with that at pH 7.0, if Pi is omitted from the binding assay. 6. Cerebellar and adrenal microsomes were used in a ligand-displacement mass assay (conducted under near-physiological conditions, at pH 7.0) on extracts of cerebral-cortex slices stimulated with agonists, and both preparations faithfully detected the increases in Ins(1,3,4,5)P4 that occurred, implying that Ins(1,3,4,5)P4 is the principal ligand on these binding sites in intact cells. 7. Apparent contradictions in the literature with regard to Ins(1,3,4,5)P4 binding sites in neuronal and peripheral tissues can be largely accounted for by the data, and the properties of the binding sites detected at physiological pH are consistent with the possibility that they are putative receptors for the proposed second-messenger role for Ins(1,3,4,5)P4.

Specificity of the purified inositol (1,3,4,5) tetrakisphosphate‐binding protein from porcine platelets

“…Overall we can conclude that a more detailed study of the properties of the purified platelet Ins(l,3,4,5)P4-binding protein has emphasised its specificity under physiological conditions, and so has increased rather than decreased the strength of our interpretation [3,4,9] that this protein is a reasonable candidate to be a physiological receptor for Ins(1,3,4,5)P4. As discussed in [4], although the current data support Ins(l,3,4,5)P4 as the most likely ligand, we cannot yet rule out PtdIns(3,4,5)P3 as an alternative physiological target for this protein, and the high specificity shown here for what is the head-group of that lipid is consistent also with that possibility.…”

“…We have shown before [9] that effects ofpH on Ins(1,3,4,5)P4 binding sites vary somewhat between tissues, and are also changed by the presence of 50 mM inorganic phosphate (or, put the way that we described it [9], the effect of inorganic phosphate is different at pH 5.5 vs. pH 7.0). We therefore examined (in the absence of inorganic phosphate) the effect of pH on the purified putative Ins(1,3,4,5)P4 receptor, partly to document the behaviour of this particular non-neuronal protein, but also to compare with the behaviour of three neuronal Ins(1,3,4,5)Paspecific binding proteins studied by Theibert et al [10] using an InsP 4 affinity probe.…”

“…The latter study [9] was inevitably compromised, as are all such studies, by the omission of Mg 2+ from and inclusion of EDTA in the binding assay -essential to avoid degradation of the ligand by Mg2+-dependent phosphatases. It is entirely possible that Mg z+ might alter significantly the affinity or specificity of Ins(1,3,4,5)P4 binding, and thus would alter our interpretation of these studies namely, that Ins(1,3,4,5)P 4 is the most likely natural ligand for these binding sites.…”

“…We have earlier emphasised how Ins(1,3,4,5)P4 binding sites on membranes can be used to assay the mass of Ins (1,3,4,5)P4 in unfractionated tissue extracts under both artificial [7,8] and nearer physiological [9] conditions. The latter study [9] was inevitably compromised, as are all such studies, by the omission of Mg 2+ from and inclusion of EDTA in the binding assay -essential to avoid degradation of the ligand by Mg2+-dependent phosphatases.…”

Binding sites for α‐trinositol (inositol 1,2,6‐trisphosphate) in porcine tissues; comparison with Ins(1,4,5)P3 and Ins(1,3,4,5)P4‐binding sites

“…We checked for the alledged optimum binding at a pH close to a physiological value and at salt concentrations mimicking the intracellular milieu (Cullen & Irvine, 1992). For these reasons, buffers B and C (Cullen & Irvine, 1992) were employed as well as buffer A, found optimal by Donie etal. (1990) and by Walsh etal.…”