Stephen C. Tovey

IP3 Receptors: Toward Understanding Their Activation

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and their relatives, ryanodine receptors, are the channels that most often mediate Ca(2+) release from intracellular stores. Their regulation by Ca(2+) allows them also to propagate cytosolic Ca(2+) signals regeneratively. This brief review addresses the structural basis of IP(3)R activation by IP(3) and Ca(2+). IP(3) initiates IP(3)R activation by promoting Ca(2+) binding to a stimulatory Ca(2+)-binding site, the identity of which is unresolved. We suggest that interactions of critical phosphate groups in IP(3) with opposite sides of the clam-like IP(3)-binding core cause it to close and propagate a conformational change toward the pore via the adjacent N-terminal suppressor domain. The pore, assembled from the last pair of transmembrane domains and the intervening pore loop from each of the four IP(3)R subunits, forms a structure in which a luminal selectivity filter and a gate at the cytosolic end of the pore control cation fluxes through the IP(3)R.

Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP

Interactions between cyclic adenosine monophosphate (cAMP) and Ca2+ are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP3R) to IP3. We show that PTH communicates with IP3R via “cAMP junctions” that allow local delivery of a supramaximal concentration of cAMP to IP3R, directly increasing their sensitivity to IP3. These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP3R, but IP3R2 and AC6 are specifically associated, and inhibition of AC6 or IP3R2 expression by small interfering RNA selectively attenuates potentiation of Ca2+ signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP3R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum.

Summary Most intracellular Ca2+ signals result from opening of Ca2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca2+ into mitochondria. Ca2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca2+ signalling, but the role of lysosomal Ca2+ uptake in Ca2+ signalling is unexplored. Inhibition of lysosomal Ca2+ uptake by dissipating the H+ gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation. Bafilomycin A1 amplified the Ca2+ signals evoked by photolysis of caged Ins(1,4,5)P3 or by inhibition of ER Ca2+ pumps, and it slowed recovery from them. Ca2+ signals evoked by store-operated Ca2+ entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca2+ released by Ins(1,4,5)P3 receptors.

Synthetic partial agonists reveal key steps in IP3 receptor activation.

Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2+ channels. IP3binding to the IP3-binding core (IBC) near the N-terminal initiates conformational changes that lead to opening of a pore. The mechanisms are unresolved. We synthesized 2-O-modified IP3 analogues that are partial agonists of IP3R. These are like IP3 in their interactions with the IBC, but they are less effective than IP3 in rearranging the relationship between the IBC and N-terminal suppressor domain (SD), and they open the channel at slower rates. IP3R with a mutation in the SD occupying a position similar to the 2-O-substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or SD therefore block obligatory coupling of the IBC and SD. Analysis of ΔG for ligand binding shows that IP3 is recognised by the IBC and conformational changes then propagate entirely via the SD to the pore.

Reliable encoding of stimulus intensities within random sequences of intracellular Ca2+ spikes.

Mathematical analysis of Ca2+ signals in single cells reveals how cells can encode stimulus intensity in the frequency of Ca2+ spikes. Apparently Random Signals Encode Information Reliably Repetitive Ca2+ spikes occur in many cells in response to stimuli that activate an intracellular signaling cascade that involves Ca2+ released from internal stores. These repetitive spikes are believed to represent the intensity of the stimulus, such that increasing the stimulus increases the frequency of the spikes. But the time between spikes (interspike interval) is random within a cell, and cells in a population exhibit variable spiking frequencies. Thurley et al. performed single-cell Ca2+ imaging of primary liver cells and human embryonic kidney (HEK) 293 cells to examine the properties of Ca2+ spikes in response to extracellular ligands under various conditions. Mathematical analysis revealed that, although the interspike interval had a random element, there was a consistent fold change in this interval across populations of cells responding to different amounts of the ligands. Thus, a common change to a random element enables the cells to properly interpret signal intensity from the frequency of repetitive Ca2+ spikes. Ca2+ is a ubiquitous intracellular messenger that regulates diverse cellular activities. Extracellular stimuli often evoke sequences of intracellular Ca2+ spikes, and spike frequency may encode stimulus intensity. However, the timing of spikes within a cell is random because each interspike interval has a large stochastic component. In human embryonic kidney (HEK) 293 cells and rat primary hepatocytes, we found that the average interspike interval also varied between individual cells. To evaluate how individual cells reliably encoded stimuli when Ca2+ spikes exhibited such unpredictability, we combined Ca2+ imaging of single cells with mathematical analyses of the Ca2+ spikes evoked by receptors that stimulate formation of inositol 1,4,5-trisphosphate (IP3). This analysis revealed that signal-to-noise ratios were improved by slow recovery from feedback inhibition of Ca2+ spiking operating at the whole-cell level and that they were robust against perturbations of the signaling pathway. Despite variability in the frequency of Ca2+ spikes between cells, steps in stimulus intensity caused the stochastic period of the interspike interval to change by the same factor in all cells. These fold changes reliably encoded changes in stimulus intensity, and they resulted in an exponential dependence of average interspike interval on stimulation strength. We conclude that Ca2+ spikes enable reliable signaling in a cell population despite randomness and cell-to-cell variability, because global feedback reduces noise, and changes in stimulus intensity are represented by fold changes in the stochastic period of the interspike interval.

Regulation of Inositol 1,4,5-Trisphosphate Receptors by cAMP Independent of cAMP-dependent Protein Kinase

In HEK cells stably expressing type 1 receptors for parathyroid hormone (PTH), PTH causes a sensitization of inositol 1,4,5-trisphosphate receptors (IP3R) to IP3 that is entirely mediated by cAMP and requires cAMP to pass directly from type 6 adenylyl cyclase (AC6) to IP3R2. Using DT40 cells expressing single subtypes of mammalian IP3R, we demonstrate that high concentrations of cAMP similarly sensitize all IP3R isoforms to IP3 by a mechanism that does not require cAMP-dependent protein kinase (PKA). IP3 binding to IP3R2 is unaffected by cAMP, and sensitization is not mediated by the site through which ATP potentiates responses to IP3. In single channel recordings from excised nuclear patches of cells expressing IP3R2, cAMP alone had no effect, but it increased the open probability of IP3R2 activated by a submaximal concentration of IP3 alone or in combination with a maximally effective concentration of ATP. These results establish that cAMP itself increases the sensitivity of all IP3R subtypes to IP3. For IP3R2, this sensitization results from cAMP binding to a novel site that increases the efficacy of IP3. Using stably expressed short hairpin RNA to reduce expression of the G-protein, Gαs, we demonstrate that attenuation of AC activity by loss of Gαs more substantially reduces sensitization of IP3R by PTH than does comparable direct inhibition of AC. This suggests that Gαs may also specifically associate with each AC·IP3R complex. We conclude that all three subtypes of IP3R are regulated by cAMP independent of PKA. In HEK cells, where IP3R2 selectively associates with AC6, Gαs also associates with the AC·IP3R signaling junction.

Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor

Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Interactions of the commonly used antagonists of IP3Rs with IP3R subtypes are poorly understood.

IP3 receptors: some lessons from DT40 cells.

Summary:  Inositol‐1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that are regulated by IP3 and Ca2+ and are modulated by many additional signals. These properties allow them to initiate and, via Ca2+‐induced Ca2+ release, regeneratively propagate Ca2+ signals evoked by receptors that stimulate formation of IP3. The ubiquitous expression of IP3R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP3R. DT40 cells are a pre‐B‐lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40‐knockout cells with both alleles of each of the three IP3R genes disrupted provide the only null‐background for analysis of homogenous recombinant IP3R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP3R behavior. Patch‐clamp recording from the nuclear envelope and Ca2+ release from intracellular stores loaded with a low‐affinity Ca2+ indicator address the mechanisms leading to activation of IP3R. We show that IP3 causes intracellular IP3R to cluster and re‐tune their responses to IP3 and Ca2+, better equipping them to mediate regenerative Ca2+ signals. Finally, we show that DT40 cells reliably count very few IP3R into the plasma membrane, where they mediate about half the Ca2+ entry evoked by the B‐cell antigen receptor.

Rapid functional assays of intracellular Ca2+ channels.

Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs. A low-affinity Ca2+ indicator (mag-fluo-4) trapped within the ER of permeabilized cells is shown to report changes in luminal free Ca2+ concentration reliably. An automated fluorescence plate reader allows rapid measurement of Ca2+ release from intracellular stores mediated by IP3R. The method can be readily adapted to other cell types or to the analysis of other intracellular Ca2+ channels. This protocol can be completed in 2–3 h.

Timescales of IP3-Evoked Ca2+ Spikes Emerge from Ca2+ Puffs Only at the Cellular Level

The behavior of biological systems is determined by the properties of their component molecules, but the interactions are usually too complex to understand fully how molecular behavior generates cellular behavior. Ca(2+) signaling by inositol trisphosphate receptors (IP(3)R) offers an opportunity to understand this relationship because the cellular behavior is defined largely by Ca(2+)-mediated interactions between IP(3)R. Ca(2+) released by a cluster of IP(3)R (giving a local Ca(2+) puff) diffuses and ignites the behavior of neighboring clusters (to give repetitive global Ca(2+) spikes). We use total internal reflection fluorescence microscopy of two mammalian cell lines to define the temporal relationships between Ca(2+) puffs (interpuff intervals, IPI) and Ca(2+) spikes (interspike intervals) evoked by flash photolysis of caged IP(3). We find that IPI are much shorter than interspike intervals, that puff activity is stochastic with a recovery time that is much shorter than the refractory period of the cell, and that IPI are not periodic. We conclude that Ca(2+) spikes do not arise from oscillatory dynamics of IP(3)R clusters, but that repetitive Ca(2+) spiking with its longer timescales is an emergent property of the dynamics of the whole cluster array.