Experimental and theoretical study of IBC domain from human IP3R2; molecular cloning, bacterial expression and protein purification.

IP3 is a ubiquitous second messenger in eukaryotic cells that triggers Ca2+-release from intracellular stores. IP3 binds to intracellular IP3-receptor (IP3R) and induces conformational change within the ligand-binding domain which regulates Ca2+-release; hence, both IP3 and IP3R are key components of the signal transduction mechanism. Here we present cDNA cloning of IP3-binding core (IBC) domain encoding only residues 224-604 of human IP3R type 2 that binds to IP3 with high affinity. RNA extraction, RT-PCR, PCR and cloning were carried out, and then the cloned DNA was checked by sequencing. Thereafter, expression vector pET-28a harboring the correct gene was transformed into different E. coli (DE3) strains and investigated its protein expression under various conditions. Finally, the IBC expression was induced at 20 °C for 20 h into BL21 strain at LB medium with 4 mM lactose and 0.5 mM IPTG, and then confirmed by western blotting. After protein purification, structural study was recorded in absence and presence of its ligand. Far-CD and intrinsic fluorescence spectra analysis of the purified protein with and without IP3 ligand showed change in secondary and tertiary IBC structure. Moreover, bioinformatics study demonstrated that the ligand binding site residues R269, K508 and R511 are conserved.

Protein complex immunological separation assay (ProCISA): a technique for investigating single protein properties.

Analysis of the posttranslational modification of proteins, such as phosphorylation, might yield misleading results due to the presence of other proteins with similar electrophoretic properties that coimmunoprecipitate with the target protein. The aim of the present work was to develop a reliable, easy and economical technique to completely isolate a protein from its complex. Here we present a new assay developed to fully isolate proteins from macromolecular complexes that consists of an initial SDS/PAGE (under reducing conditions), which isolates the target protein, followed by transfer of the proteins to a buffer, from which the target protein is recaptured by conventional immunoprecipitation. This technique, that we have termed "Protein Complex Immunological Separation Assay" (ProCISA), successfully separated proteins of different sizes, such as pp60Src and the IP3 receptor (IP3R), from their complexes. We show that ProCISA allows the investigation of the tyrosine phosphorylation state of isolated proteins. This technique could also be used to study other posttranslational modifications without risk of misleading results resulting from contamination with other proteins of similar electrophoretic mobility which complex with the protein of interest.

Reconstitution of endoplasmic reticulum InsP3 receptors into black lipid membranes.

Intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca(2+)) channels, are most often studied through their reconstitution into planar lipid bilayers (also called black lipid membranes, or BLMs). General methods for making bilayers and for ion channel reconstitution into BLMs have been extensively detailed elsewhere; thus, here the focus is on specific details relevant for inositol(1,4,5)-trisphosphate receptor (InsP3R) recordings. These procedures describe how to perform single-channel recordings of native or recombinant InsP3Rs in BLMs. Similar procedures are used to study native or recombinant ryanodine receptors (RyanRs) in BLMs.

Characterization of a flatworm inositol (1,4,5) trisphosphate receptor (IP3R) reveals a role in reproductive physiology.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca²âº channels that elevate cytoplasmic Ca²âº in response to the second messenger IP3. Here, we describe the identification and in vivo functional characterization of the planarian IP3R, the first intracellular Ca²âº channel to be defined in flatworms. A single IP3R gene in Dugesia japonica encoded a 2666 amino acid protein (Dj.IP3R) that shared well conserved structural features with vertebrate IP3R counterparts. Expression of an NH2-terminal Dj.IP3R region (amino acid residues 223-585) recovered high affinity ³H-IP3 binding (0.9±0.1 nM) which was abolished by a single point mutation of an arginine residue (R495L) important for IP3 coordination. In situ hybridization revealed that Dj.IP3R mRNA was most strongly expressed in the pharynx and optical nerve system as well as the reproductive system in sexualized planarians. Consistent with this observed tissue distribution, in vivo RNAi of Dj.IP3R resulted in a decreased egg-laying behavior suggesting Dj.IP3R plays an upstream role in planarian reproductive physiology.

Flexible architecture of IP3R1 by Cryo-EM.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.

Isolation of inositol 1,4,5-trisphosphate receptor-associating proteins and selective knockdown using RNA interference.

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are IP(3)-gated Ca(2+) release channels localized on intracellular Ca(2+) stores and play a role in the generation of complex patterns of intracellular Ca(2+) signals. We show herein experimental protocols for the identification of associating proteins of IP(3)R isoforms from various cells and tissues using affinity column chromatography and for the specific knockdown of the expression of IP(3)R isoforms and their associating proteins using RNA interference. These methods will provide clues to understand the exact nature of how the signaling complex contributes to the generation of spatio-temporal patterns of intracellular Ca(2+) signals.