IP3 receptors – lessons from analyses ex cellula

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are widely expressed intracellular channels that release Ca2+ from the endoplasmic reticulum (ER). We review how studies of IP3Rs removed from their intracellular environment (‘ex cellula’), alongside similar analyses of ryanodine receptors, have contributed to understanding IP3R behaviour. Analyses of permeabilized cells demonstrated that the ER is the major intracellular Ca2+ store, and that IP3 stimulates Ca2+ release from it. Radioligand binding confirmed that the 4,5-phosphates of IP3 are essential for activating IP3Rs, and facilitated IP3R purification and cloning, which paved the way to structural analyses. Reconstitution of IP3Rs into lipid bilayers and patch-clamp recording from the nuclear envelope established that IP3Rs have a large conductance and select weakly between Ca2+ and other cations. Structural analyses are now revealing how IP3 binding to the N-terminus of the tetrameric IP3R opens the pore 7nm away from the IP3-binding core (IBC). Communication between the IBC and pore passes through a nexus of interleaved domains contributed by structures associated with the pore and cytosolic domains, which together contribute to a Ca2+-binding site. These structural analyses provide a plausible explanation for the suggestion that IP3 gates IP3Rs by first stimulating Ca2+ binding, which leads to pore opening and Ca2+ release

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells

Endogenous signalling pathways and caged-IP3 evoke Ca2+ puffs at the same abundant immobile intracellular sites

The building blocks for intracellular Ca2+ signals evoked by inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ puffs, transient focal increases in Ca2+ concentration that reflect the opening of a small clusters of IP3Rs. We use total internal reflection fluorescence microscopy and automated analyses to detect Ca2+ puffs in human embryonic kidney 293 cells evoked by photolysis of caged-IP3 or activation of endogenous muscarinic receptors with carbachol. Ca2+ puffs evoked by carbachol initiated at an estimated 65 ± 7 sites/cell, and the sites remained immobile for many minutes. Photolysis of caged-IP3 evoked Ca2+ puffs at a similar number of sites (100 ± 35). Increasing the carbachol concentration increased the frequency of Ca2+ puffs without unmasking additional Ca2+ release sites. By measuring responses to sequential challenges with carbachol and photolysis of caged-IP3, we established that the two stimuli evoked Ca2+ puffs at the same sites. We conclude that IP3-evoked Ca2+ puffs initiate at numerous immobile sites, the sites become more likely to fire as the IP3 concentration increases, and there is no evidence that endogenous signalling pathways selectively deliver IP3 to specific sites.This work was supported by the Wellcome Trust [grant number 101844], by a studentship to M.V.K. from the Cambridge Overseas Trust, and by awards to M.V.K. from St. John's College, Cambridge and the Cambridge Philosophical Society. Deposited in PMC for immediate release

Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A

In human aortic smooth muscle cells (ASMC), prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration ([Ca2+]i) evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP (EPACs) were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to selectively activate EP2 receptors), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through ‘hyperactive signalling junctions’, wherein cAMP is locally delivered to PKA at super-saturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signalling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals.This work was supported by the Medical Research Council [G0900049], Biotechnology and Biological Sciences Research Council [L000075] and the Wellcome Trust [101844]

Three-dimensional structure of recombinant type 1 inositol 1,4,5-trisphosphate receptor.

IP3Rs (inositol 1,4,5-trisphosphate receptors) are the intracellular channels that mediate release of Ca2+ from the endoplasmic reticulum in response to the many stimuli that evoke Ins(1,4,5)P3 formation. We characterized and purified type 1 IP3R heterologously expressed in Sf9 insect cells, and used the purified IP3R1 to determine its three-dimensional structure by electron microscopy and single-particle analysis. Recombinant IP3R1 has 4-fold symmetry with overall dimensions of approx. 19.5 nm x 19.5 nm x 17.5 nm. It comprises a small domain, which is likely to include the pore, linked by slender bridges to a large cytoplasmic domain with four petal-like regions. Our structures of recombinant IP3R1 and native cerebellar IP3R have similar appearances and dimensions. The only notable difference is the absence of a central stigma-like domain from the cytoplasmic region of recombinant IP3R1. The first structure of a recombinant IP3R is an important step towards developing three-dimensional structures of IP3R that better contribute to our understanding of the structural basis of IP3R activation

Immobile IP3 Receptor Clusters: Building Blocks For IP3-Evoked Ca2+ Signals

Co-regulation of IP3 receptors (IP3Rs) by IP3 and cytosolic Ca2+ allows them to mediate regenerative signals, amongst which are Ca2+ puffs. These reflect the near-simultaneous opening of a few IP3Rs within a small cluster. A long-standing conundrum is the observation that while most IP3Rs appear to be mobile, Ca2+ puffs repeatedly initiate from a limited number of fixed sites. Using gene-editing to attach GFP to endogenous IP3Rs in HeLa cells has allowed the distribution of IP3Rs and the Ca2+ signals they evoke to be imaged simultaneously. This approach shows that most endogenous IP3Rs are loosely assembled into small clusters, most of which are mobile. However, the Ca2+ puffs evoked by histamine or photolysis of caged IP3 invariably initiated at immobile IP3R clusters adjacent to the plasma membrane (PM). Hence, only a small fraction of cellular IP3Rs are 'licensed' to respond. The licensed IP3R clusters sit alongside the sites where store-operated Ca2+ entry (SOCE) occurs, suggesting that the IP3Rs may allow local regulation of SOCE

Hepatitis C treatment: where are we now?

Chronic hepatitis C infection affects millions of people worldwide and confers significant morbidity and mortality. Effective treatment is needed to prevent disease progression and associated complications. Previous treatment options were limited to interferon and ribavirin regimens, which gave low cure rates and were associated with unpleasant side effects. The era of direct acting antiviral (DAA) therapies began with the development of the first-generation of NS3/4A protease inhibitors (PI) in 2011. They vastly improved outcomes for patients, particularly those with genotype 1 infection, the most prevalent genotype globally. Since then a multitude of DAAs have been licensed for use and outcomes for patients have improved further, with fewer side effects and cure rates approaching 100%. Recent regimens are interferon-free, and in many cases, ribavirin-free and involve a combination of DAA agents. This review summarises the treatment options currently available and discusses potential barriers that may delay the global eradication of hepatitis C

The effect of intervertebral cartilage on neutral posture and range of motion in the necks of sauropod dinosaurs

The necks of sauropod dinosaurs were a key factor in their evolution. The habitual posture and range of motion of these necks has been controversial, and computer-aided studies have argued for an obligatory sub-horizontal pose. However, such studies are compromised by their failure to take into account the important role of intervertebral cartilage. This cartilage takes very different forms in different animals. Mammals and crocodilians have intervertebral discs, while birds have synovial joints in their necks. The form and thickness of cartilage varies significantly even among closely related taxa. We cannot yet tell whether the neck joints of sauropods more closely resembled those of birds or mammals. Inspection of CT scans showed cartilage:bone ratios of 4.5% for Sauroposeidon and about 20% and 15% for two juvenile Apatosaurus individuals. In extant animals, this ratio varied from 2.59% for the rhea to 24% for a juvenile giraffe. It is not yet possible to disentangle ontogenetic and taxonomic signals, but mammal cartilage is generally three times as thick as that of birds. Our most detailed work, on a turkey, yielded a cartilage:bone ratio of 4.56%. Articular cartilage also added 11% to the length of the turkey's zygapophyseal facets. Simple image manipulation suggests that incorporating 4.56% of neck cartilage into an intervertebral joint of a turkey raises neutral posture by 15°. If this were also true of sauropods, the true neutral pose of the neck would be much higher than has been depicted. An additional 11% of zygapophyseal facet length translates to 11% more range of motion at each joint. More precise quantitative results must await detailed modelling. In summary, including cartilage in our models of sauropod necks shows that they were longer, more elevated and more flexible than previously recognised