Analysis of IP3 receptors in and out of cells

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP₃R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca^2 + channels.

Scope of the review

In this brief review, we first consider a variety of complementary methods that allow the links between IP₃ binding and channel gating to be defined. How does IP₃ binding to the IP₃-binding core in each IP₃R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP₃, Ca^2 + signals and IP₃R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP₃R signalling.

Major conclusions

All IP₃R are regulated by both IP₃ and Ca^2 +. This allows them to initiate and regeneratively propagate intracellular Ca^2 + signals. The elementary Ca^2 + release events evoked by IP₃ in intact cells are mediated by very small numbers of active IP₃R and the Ca^2 +-mediated interactions between them. The spatial organization of these Ca^2 + signals and their stochastic dependence on so few IP₃Rs highlight the need for methods that allow the spatial organization of IP₃R signalling to be addressed with single-molecule resolution.

General significance

A variety of complementary methods provide insight into the structural basis of IP₃R activation and the contributions of IP₃-evoked Ca^2 + signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Dynamic clustering of IP3 receptors by IP3

The versatility of Ca2+ as an intracellular messenger stems largely from the impressive, but complex, spatiotemporal organization of the Ca2+ signals. For example, the latter when initiated by IP3 (inositol 1,4,5-trisphosphate) in many cells manifest hierarchical recruitment of elementary Ca2+ release events ('blips' and then 'puffs') en route to global regenerative Ca2+ waves as the cellular IP3 concentration rises. The spacing of IP3Rs (IP3 receptors) and their regulation by Ca2+ are key determinants of these spatially organized Ca2+ signals, but neither is adequately understood. IP3Rs have been proposed to be pre-assembled into clusters, but their composition, geometry and whether clustering affects IP3R behaviour are unknown. Using patch-clamp recording from the outer nuclear envelope of DT40 cells expressing rat IP3R1 or IP3R3, we have recently shown that low concentrations of IP3 cause IP3Rs to aggregate rapidly and reversibly into small clusters of approximately four IP3Rs. At resting cytosolic Ca2+ concentrations, clustered IP3Rs open independently, but with lower open probability, shorter open duration and lesser IP3-sensitivity than lone IP3Rs. This inhibitory influence of clustering on IP3R is reversed when the [Ca2+]i (cytosolic free Ca2+ concentration) increases. The gating of clustered IP3Rs exposed to increased [Ca2+]i is coupled: they are more likely to open and close together, and their simultaneous openings are prolonged. Dynamic clustering of IP3Rs by IP3 thus exposes them to local Ca2+ rises and increases their propensity for a CICR (Ca2+-induced Ca2+ rise), thereby facilitating hierarchical recruitment of the elementary events that underlie all IP3-evoked Ca2+ signals.     

Interplay between ryanodine and IP3 receptors in ATP-stimulated mouse luteinized-granulosa cells

In mouse luteinized-granulosa cells (MGLC), ATP induces an increase in intracellular Ca2+ concentration by stimulating phospholipase C (PLC) associated with purinergic receptors, leading to production of inositol 1,4,5-trisphosphate (IP3) and subsequent release of Ca2+ from intracellular stores. In this study, we examined the cross-talk between the ryanodine receptors (RyR) and IP3 receptors (IP3R) in response to ATP in MGLC. Specifically, the effect of RyR modulators on ATP response was examined. The results showed that ATP-induced intracellular calcium elevation was abolished by inhibitors of the RyR, such as dantrolene (25 microM) and ryanodine (80 microM). When the MGLC were stimulated with activators of RyR, 2 microM ryanodine and 10 mM caffeine, the ATP-elicited response was decreased. These actions were independent of IP3 production stimulated by ATP. Hence, ATP-induced intracellular Ca2+ mobilization involves the coordinated action of both types of calcium release channels (CRCs). Using fluorescent probes, it was shown that IP3R is uniformly distributed throughout the cell; in contrast, RyR is mainly found around the nuclei. It is concluded that the IP3R and the RyR are functionally associated, and both play a role in the pattern of Ca2+ increase observed during purinergic stimulation of MGLC. This coupling may provide a highly efficient amplification mechanism for ATP stimulation of Ca2+ mobilization.     

Structure and function of IP3 receptors

The Molecular, structural and functional characteristics of the intracellular Ca²⁺ release channel activated by inositol 1,4,5-trisphosphate (IP₃), also named IP₃ receptor (IP₃R), are described here. We also discuss the differences in primary structure, expression and modulation of the receptor subtypes and their physiological roles. The similarity and differences between the IP₃R and the other intracellular Ca²⁺ channel, the ryanodine receptor, are briefly presented.     

Type 3 IP3 receptors driving oncogenesis

Type 3 Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R3s) have been identified as anti-oncogenic channels by propelling pro-apoptotic Ca²⁺ signals to mitochondria. Yet, recent studies (Rezuchova et al, Cell Death Dis, 2019; Ueasilamongkol et al, Hepathology, 2019; Guerra et al, Gut, 2019) revealed that IP₃R3 upregulation drives oncogenesis via ER-mitochondrial Ca²⁺ crosstalk, adding complexity to IP₃R3’s role in cancer.     

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.     

IP3 receptors and Ca2+ signals in adult skeletal muscle satellite cells in situ

In this short article we review muscle satellite cell characteristics and our studies in adult rodent muscle satellite cells in situ. Using confocal laser scanning microscopy and immunocytochemistry, a high level of IP3 receptor (IP3R) immunostaining was detected in satellite cells. These cells were identified by their peripheral position, their size, the shape of their nucleus, the paucity of the apparent cytoplasm, and the immunostaining with specific molecular markers such as alpha-actinin, the neural cell adhesion molecule (N-CAM) and desmin. High extracellular K+ (60 mM) induced long-lasting Ca2+ signals in satellite cells in situ. We suggest that electrical activity stimulates IP3-associated Ca2+ signals that could act in concert with signaling pathways triggered by growth factors and/or hormones.     

Rapid functional assays of recombinant IP3 receptors

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).     

Molecular and binding characteristics of IP3 receptors in bovine spermatozoa

We have shown the presence of inositol 1,4,5-triphosphate (IP3) receptors in bovine spermatozoa. These receptors are mainly localized and functionally associated with the acrosome region. Molecular characterization of these bovine IP3 receptors has shown that the functional size of the IP3 binding domain is a protein of 66+/-2 kDa, in agreement with the size of both bovine adrenal cortex and bovine adrenal medullar chromaffin cells IP3 receptors. In contrast, bovine cerebellum IP3 receptor displays molecular weight of 220+/-5 kDa, a value in agreement with data in the literature. Bovine IP3 receptors have a one-affinity state characterized by a low affinity (Kd 750 nM) and a relatively high density (7.5 pmol/mg protein). They are functional and release internal calcium upon the binding of the second messenger. Moreover, the finding that the specific A1 adenosine receptor agonist R-PIA elicits almost the same effect as IP3 might be of some help in understanding the physiological role of these inhibitory adenosine receptors in mammalian spermatozoa.     

IP(3) receptors, stress and apoptosis

Inositol 1,4,5-trisphosphate (IP3) receptors are intracellular calcium channels that are able to release calcium from intracellular stores upon activation by IP3 and modulation by calcium. IP3 receptors are involved in variety of processes during physiological, but also in the pathophysiological states. Unraveling their regulation and function, especially under the pathological situations can result in a development of new therapeutic strategies based on the IP3 receptor′s activation and/or blocking. To the stimuli that can modulate IP3 receptors belong several stress factors (e.g. immobilization stress, oxidative stress and hypoxia) and also apoptosis. Depending on the length and strength of the stress stimulus, expression of IP3 receptors can be increased, or decreased. Therefore, in this minireview modulation of IP3 receptors by some stressors is discussed. Since it was already shown that strong hypoxia might lead to the apoptosis induction, special focus will be given to the hypoxic stress and induction of apoptosis.     

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.     

Cellular geography of IP3 receptors, STIM and Orai: a lesson from secretory epithelial cells

Pancreatic acinar cells exhibit a remarkable polarization of Ca2+ release and Ca2+ influx mechanisms. In the present brief review, we discuss the localization of channels responsible for Ca2+ release [mainly IP3 (inositol 1,4,5-trisphosphate) receptors] and proteins responsible for SOCE (store-operated Ca2+ entry). We also place these Ca2+-transporting mechanisms on the map of cellular organelles in pancreatic acinar cells, and discuss the physiological implications of the cellular geography of Ca2+ signalling. Finally, we highlight some unresolved questions stemming from recent observations of co-localization and co-immunoprecipitation of IP3 receptors with Orai channels in the apical (secretory) region of pancreatic acinar cells.     

IP3 type 1 receptors in the heart: their predominance in atrial walls with ganglion cells

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

When worlds collide: IP3 receptors and the ERAD pathway

While cell signaling devotees tend to think of the endoplasmic reticulum (ER) as a Ca²⁺ store, those who study protein synthesis tend to see it more as site for protein maturation, or even degradation when proteins do not fold properly. These two worldviews collide when inositol 1,4,5-trisphosphate (IP₃) receptors are activated, since in addition to acting as release channels for stored ER Ca²⁺, IP₃ receptors are rapidly destroyed via the ER-associated degradation (ERAD) pathway, a ubiquitination- and proteasome-dependent mechanism that clears the ER of aberrant proteins. Here we review recent studies showing that activated IP₃ receptors are ubiquitinated in an unexpectedly complex manner, and that a novel complex composed of the ER membrane proteins SPFH1 and SPFH2 (erlin 1 and 2) binds to IP₃ receptors immediately after they are activated and mediates their ERAD. Remarkably, it seems that the conformational changes that underpin channel opening make IP₃ receptors resemble aberrant proteins, which triggers their binding to the SPFH1/2 complex, their ubiquitination and extraction from the ER membrane and finally, their degradation by the proteasome. This degradation of activated IP₃ receptors by the ERAD pathway serves to reduce the sensitivity of ER Ca²⁺ stores to IP₃ and may protect cells against deleterious effects of over-activation of Ca²⁺ signaling pathways.     

ATP and adenosine trigger the interaction of plasma membrane IP3 receptors with protein kinase A in oviductal ciliated cells

We have demonstrated that adenosine did not produce any change of intracellular free Ca²⁺ concentration ([Ca²⁺]i) in oviductal ciliated cells; however, it increased the ATP-induced Ca²⁺ influx through the activation of protein kinase A (PKA). Uncaging of IP₃ and cAMP triggered a larger Ca²⁺ influx than did IP₃ alone. Furthermore, the IP₃ effect was abolished by Xestospongin C, an IP₃ receptor blocker. Whole-cell recordings demonstrated the presence of an ATP-induced Ca²⁺ current, and the addition of adenosine increased the peak of this current. This effect was not observed in the presence of H-89, a PKA inhibitor. Using excised macro-patches of plasma membrane, IP₃ generated a current, which was higher in the presence of the catalytic PKA subunit and this current was blocked by Xestospongin C. We show here that activation of plasma membrane IP₃ receptors directly triggers Ca²⁺ influx in response to ATP and that these receptors are modulated by adenosine-activated PKA.