Disease-associated mutations in inositol 1,4,5-trisphosphate receptor subunits impair channel function.

The inositol 1,4,5-trisphosphate (IP) receptors (IPRs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IPRs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IPR function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized.

IP receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer.

Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts.

Bcl-2 and IP compete for the ligand-binding domain of IPRs modulating Ca signaling output.

Bcl-2 proteins have emerged as critical regulators of intracellular Ca dynamics by directly targeting and inhibiting the IP receptor (IPR), a major intracellular Ca-release channel. Here, we demonstrate that such inhibition occurs under conditions of basal, but not high IPR activity, since overexpressed and purified Bcl-2 (or its BH4 domain) can inhibit IPR function provoked by low concentration of agonist or IP, while fails to attenuate against high concentration of agonist or IP. Surprisingly, Bcl-2 remained capable of inhibiting IPR1 channels lacking the residues encompassing the previously identified Bcl-2-binding site (a.

All three IP receptor isoforms generate Ca puffs that display similar characteristics.

Inositol 1,4,5-trisphosphate (IP) evokes Ca release through IP receptors (IPRs) to generate both local Ca puffs arising from concerted openings of clustered IPRs and cell-wide Ca waves. Imaging Ca puffs with single-channel resolution yields information on the localization and properties of native IPRs in intact cells, but interpretation has been complicated because cells express varying proportions of three structurally and functionally distinct isoforms of IPRs. Here, we used TIRF and light-sheet microscopy to image Ca puffs in HEK-293 cell lines generated by CRISPR-Cas9 technology to express exclusively IPR type 1, 2, or 3.

Inositol 1,4,5-trisphosphate Receptor Mutations associated with Human Disease.

Calcium release into the cytosol via the inositol 1,4,5-trisphosphate receptor (IPR) calcium channel is important for a variety of cellular processes. As a result, impairment or inhibition of this release can result in disease. Recently, mutations in all four domains of the IPR have been suggested to cause diseases such as ataxia, cancer, and anhidrosis; however, most of these mutations have not been functionally characterized.

Region-specific proteolysis differentially modulates type 2 and type 3 inositol 1,4,5-trisphosphate receptor activity in models of acute pancreatitis.

Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IPR) by a diverse array of regulatory inputs results in intracellular Ca signals with distinct characteristics. These events allow the activation of specific downstream effectors. We reported previously that region-specific proteolysis represents a novel regulatory event for type 1 IPR (R1).

Region-specific proteolysis differentially regulates type 1 inositol 1,4,5-trisphosphate receptor activity.

The inositol 1,4,5 trisphosphate receptor (IPR) is an intracellular Ca release channel expressed predominately on the membranes of the endoplasmic reticulum. IPR1 can be cleaved by caspase or calpain into at least two receptor fragments. However, the functional consequences of receptor fragmentation are poorly understood.

DPB162-AE, an inhibitor of store-operated Ca entry, can deplete the endoplasmic reticulum Ca store.

Store-operated Ca entry (SOCE), an important Ca signaling pathway in non-excitable cells, regulates a variety of cellular functions. To study its physiological role, pharmacological tools, like 2-aminoethyl diphenylborinate (2-APB), are used to impact SOCE. 2-APB is one of the best characterized SOCE inhibitors.

Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca(2+)-release channels with each subunit containing a binding site for IP3in the amino terminus. We provide evidence that four IP3molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca(2+)release suggested that IP3Rs are maximally occupied by IP3before substantial Ca(2+)release occurs.

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs.

The ability of inositol 1,4,5-trisphosphate receptors (IP3R) to precisely initiate and generate a diverse variety of intracellular Ca(2+) signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP3, Ca(2+), and ATP). However, the contribution of IP3R heterotetramerization to Ca(2+) signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation.

Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors.

Using concatenated subunits to investigate the functional consequences of heterotetrameric inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60-70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP.

Tracing the Evolutionary History of Inositol, 1, 4, 5-Trisphosphate Receptor: Insights from Analyses of Capsaspora owczarzaki Ca2+ Release Channel Orthologs.

Cellular Ca(2+) homeostasis is tightly regulated and is pivotal to life. Inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) are the major ion channels that regulate Ca(2+) release from intracellular stores. Although these channels have been extensively investigated in multicellular organisms, an appreciation of their evolution and the biology of orthologs in unicellular organisms is largely lacking.

Functional inositol 1,4,5-trisphosphate receptors assembled from concatenated homo- and heteromeric subunits.

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events.

Fragmented inositol 1,4,5-trisphosphate receptors retain tetrameric architecture and form functional Ca2+ release channels.

Inositol 1,4,5-trisphosphate receptor isoforms are a family of ubiquitously expressed ligand-gated channels encoded by three individual genes. The proteins are localized to membranes of intracellular Ca(2+) stores and play pivotal roles in Ca(2+) homeostasis. Previous studies have demonstrated that IP3R1 is cleaved by the intracellular proteases calpain and caspase both in vivo and in vitro.

Mass spectrometric analysis of type 1 inositol 1,4,5-trisphosphate receptor ubiquitination.

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric channels in endoplasmic reticulum membranes of mammalian cells and mediate IP(3)-induced calcium mobilization. In response to various extracellular stimuli that persistently elevate IP(3) levels, IP(3) receptors are also ubiquitinated and then degraded by the proteasome. Here, for endogenous type 1 IP(3) receptor (IP(3)R1) activated by endogenous signaling pathways and processed by endogenous enzymes, we sought to determine the sites of ubiquitination and the composition of attached ubiquitin conjugates.

The role of Ca2+ in triggering inositol 1,4,5-trisphosphate receptor ubiquitination.

The IP3R (inositol 1,4,5-trisphosphate receptor) forms tetrameric Ca2+ channels in ER (endoplasmic reticulum) membranes, where channel activity is largely under the control of the co-agonists IP3 and Ca2+. In cells stimulated using extracellular ligands that persistently elevate phosphoinositidase C activity, IP3Rs are rapidly ubiquitinated and then degraded by the proteasome through as yet undefined mechanisms. Whereas binding of IP3 has been suggested to be a key event in the triggering of IP3R ubiquitination the role of Ca2+ in this process remains unknown.

Involvement of the p97-Ufd1-Npl4 complex in the regulated endoplasmic reticulum-associated degradation of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate (IP(3)) receptors form tetrameric, IP(3)-gated channels in endoplasmic reticulum membranes that govern the release of Ca(2+) from this organelle. In response to activation of certain G protein-coupled receptors that persistently elevate IP(3) concentration, IP(3) receptors are ubiquitinated and degraded by the ubiquitin-proteasome pathway. IP(3) receptor ubiquitination is mediated by the ubiquitin-conjugating enzyme, (mam)Ubc7, a component of the endoplasmic reticulum-associated degradation pathway.