Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles

Abstract

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.

Transport of receptors, receptor signaling complexes and ion channels via neuropeptide-secretory vesicles

“…Interestingly, we noticed the presence of several channels involved in the release of Ca 2+ from intracellular Ca 2+ stores or Ca 2+ influx via VGCCs. Release of Ca 2+ from intracellular Ca 2+ stores can be triggered by activating IP 3 Rs and ryanodine receptors [47,48]. The existence of these receptors (Supplementary information, Table S1) in LDCV membranes is consistent with the notion that LDCVs can serve as intracellular Ca 2+ stores [49,50].…”

Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated.

“…These parameters were adjusted to limit the breakdown of IP3 and the inhibition of IP3 binding by higher Ca2+ levels (Worley et al, 1987b is largely prevented by heparin, an established antagonist of IP3 receptor binding (Worley etal., 1987b) and IP3-induced calcium release (Cullen et al, 1988;Ghosh et al, 1988). The similar inositol phosphate specificity for 45Ca2' accumulation in brain slices, IP3 receptor binding (Worley et al, 1987b), and IP3 release of calcium in numerous other systems Ghosh et al, 1989;Irvine, 1989;Nahorski, 1989;Ferris et al, 1989) (Worley et al, 1987a;1989) closely resembles the IP3 sensitive pools of 45Ca2+ ( Figure 4). …”

“…Release of calcium by IP3 is mediated by a specific receptor protein which has been localized in the brain by autoradiography (Worley et al, 1987a(Worley et al, , 1989 and immunohistochemistry (Ross et al, 1989a), purified to homogeneity (Supattapone et al, 1988), molecularly cloned (Furuichi et al, 1989), and shown to comprise within a four-subunit 1000-kD protein both the IP3 recognition site and its associated calcium channel (Ferris et al, 1989). Immunohistochemical examination of cerebellar Purkinje cells at the electron microscopic level reveals IP3 receptors selectively localized to a subdivision of the ER (Ross et al, 1989a).…”

Do submaximal InsP3 concentrations only induce the partial discharge of permeabilized hepatocyte calcium pools because of the concomitant reduction of intraluminal Ca2+ concentration?

“…channel, as after its reconstitution into lipid vesicles these vesicles were able to release Ca"' in response to stimulation by lnsP3 [2]. Subsequently, however, it was suspected that the process of Ca?+ release by InsP, presented an additional complexity, since the addition of low concentrations of InsPj to permeabilized cells was found to induce the fast release of a part of the InsP3-sensitive calcium pool instead of the slow discharge of the pool [3].…”