Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric intracellular Ca2+-release channels with each subunit containing a binding site for IP3 in the N-terminus. We provide evidence that four IP3 molecules are required to activate the channel under diverse conditions. Comparing the concentration-response relationship for binding and Ca2+ release suggested that IP3Rs are maximally occupied by IP3 before substantial Ca2+ release occurs. We showed that ligand binding–deficient subunits acted in a dominant-negative manner when coexpressed with wild-type monomers in the chicken immune cell line DT40-3KO, which lacks all three genes encoding IP3R subunits, and confirmed the same effect in an IP3R-null human cell line (HEK-3KO) generated by CRISPR/Cas9 technology. Using dimeric and tetrameric concatenated IP3Rs with increasing numbers of binding-deficient subunits, we addressed the obligate ligand stoichiometry. The concatenated IP3Rs with four ligand-binding sites exhibited Ca2+ release and electrophysiological properties of native IP3Rs. However, IP3 failed to activate IP3Rs assembled from concatenated dimers consisting of one binding-competent and one binding-deficient mutant subunit. Similarly, IP3Rs containing two monomers of IP3R2short, an IP3 binding-deficient splice variant, were nonfunctional. Concatenated tetramers containing only three binding competent ligand-binding sites were nonfunctional under a wide range of activating conditions. These data provide definitive evidence that IP3-induced Ca2+ release only occurs when each IP3R monomer within the tetramer is occupied by IP3, thereby ensuring fidelity of Ca2+ release.

The primary problems associated with using straight soybean oil as a fuel in a compression ignition internal combustion engine are caused by high fuel viscosity. Transesterification of soybean oil with an alcohol provides a significant reduction in viscosity, thereby enhancing the physical properties of the renewable fuel to improve engine performance. The ethyl and methyl esters of soybean oil with commercial diesel fuel additives revealed fuel properties that compared very well with diesel fuel, with the exception of gum formation, which manifested itself in problems with the plugging of fuel filters. Engine performance using soybean ester fuels differed little from engine performance with diesel fuel. A slight power loss combined with an increase in fuel consumption were experienced with the esters, primarily because of the lower heating value of the esters than for diesel fuel. Emissions for the 2 fuels were similar, with nitrous oxide emissions higher for the esters. Measurements of engine wear and fuel‐injection system tests showed no abnormal characteristics for any of the fuels after the 200‐hr tests. Engine deposits were comparable in amount, but slightly different in color and texture, with the methyl ester engine experiencing greater carbon and varnish deposits on the pistons.

Key points• Three family members of inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) represent ubiquitously expressed intracellular Ca 2+ release channels. The activity of the channels is regulated in a concerted and inter-related fashion by InsP 3 , Ca 2+ and ATP. It is not established whether each isoform is regulated by these ligands in an identical fashion. In this study we directly compare the single channel activity of mammalian InsP 3 R-1 and InsP 3 R-2 expressed in isolation in the presence of these ligands.• An increase in activity for each isoform was mediated by a transition from a quiescent, 'parked' state to a 'drive' mode characterized by bursting activity. Ligands did not affect the single channel activity during these bursts but instead modulate the extent of bursting activity.• Kinetic analysis revealed that the regulation of the transition to bursting activity by Ca

Based upon an extensive single-channel data set, a Markov model for types I and II inositol trisphosphate receptors (IP(3)R) is developed. The model aims to represent accurately the kinetics of both receptor types of IP(3)R depending on the concentrations of inositol trisphosphate (IP(3)), adenosine trisphosphate (ATP), and intracellular calcium (Ca(2+)). In particular, the model takes into account that for some combinations of ligands the IP(3)R switches between extended periods of inactivity alternating with intervals of bursting activity (mode changes). In a first step, the inactive and active modes are modeled separately. It is found that, within modes, both receptor types are ligand-independent. In a second step, the submodels are connected by transition rates. Ligand-dependent regulation of the channel activity is achieved by modulating these transitions between active and inactive modes. As a result, a compact representation of the IP(3)R is obtained that accurately captures stochastic single-channel dynamics including mode changes in a model with six states and 10 rate constants, only two of which are ligand-dependent.

Inositol 1,4,5-trisphosphate receptors (IP 3 R) 2 are a family of intracellular release channels that play an essential role in evoking Ca 2ϩ signals triggered by the occupation of numerous types of cell-surface receptors that are coupled to enhanced inositol-lipid turnover (1). Three different IP 3 R isoforms have been identified, and most cells appear to express multiple isoforms (2, 3). IP 3 receptors have been shown to be substrates for several different protein kinases in vivo and/or in vitro. These include protein kinase A (4, 5), protein kinase G (6, 7), protein kinase C (8), CaM kinase-II (8), Fyn tyrosine kinase (9, 10) and cdk1/CyB (11). By far the best-characterized of these effects are those of protein kinases A and G. Two serine residues have been identified as potential sites of phosphorylation in the type I IP 3 R isoform (12). Mutagenesis studies in a DT40 IP 3 R expression system suggest that both protein kinases A and G act to enhance IP 3 -mediated Ca 2ϩ release (13). However, the preferred sites of phosphorylation and functional effects on IP 3 R channel function may differ in different cell types and between alternatively spliced variants (6, 13-15). The regulatory role of the other protein kinases phosphorylating IP 3 Rs are poorly understood.The serine/threonine protein kinase Akt/protein kinase B is the cellular homologue of the viral oncogene v-Akt and is activated by various growth factors and cytokines. The membrane translocation and initial activation of the enzyme is dependent on the production of 3-phosphorylated inositol lipids catalyzed by PI 3-kinase. The activated Akt kinase then phosphorylates a number of key substrates involved in the stimulation of intermediary metabolism and promotion of cell survival, proliferation, and growth (reviewed in ). An examination of the sequence of all three IP 3 R isoforms indicates the presence of a consensus RXRXX(S/T) sequence for phosphorylation by Akt kinase (19). In the present study, we have shown for the first time that IP 3 Rs are substrates for activated Akt kinase in vivo and have investigated several possible functional consequences of this phosphorylation on Ca 2ϩ signaling.