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.     

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.     

Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a Ca(2+) release channel localized on the endoplasmic reticulum (ER) and plays an important role in neuronal function. IP(3) receptor was discovered as a developmentally regulated protein missing in the cerebellar mutant mice. Recent studies indicate that IP(3)Rs are involved in early development and neuronal plasticity. IP(3) works to release IRBIT from the IP(3) binding core in addition to release Ca(2+). IRBIT binds to and activates Na, Bicarbonate cotransporter. Electron microscopic study show the IP(3) receptor has allosteric property to change its form from square to windmill in the presence of Ca(2+). IP(3)R associates with ERp44, a redox sensor, Homer, other proteins and is transported as vesicular ER on microtubules. All these data suggests IP(3) receptor/CA(2+) channel works as a signaling center inside cells.     

Heterogeneous function of ryanodine receptors, but not IP3 receptors, in hamster cremaster muscle feed arteries and arterioles

The roles played by ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP?Rs) in vascular smooth muscle in the microcirculation remain unclear. Therefore, the function of both RyRs and IP?Rs in Ca(2+) signals and myogenic tone in hamster cremaster muscle feed arteries and downstream arterioles were assessed using confocal imaging and pressure myography. Feed artery vascular smooth muscle displayed Ca(2+) sparks and Ca(2+) waves, which were inhibited by the RyR antagonists ryanodine (10 μM) or tetracaine (100 μM). Despite the inhibition of sparks and waves, ryanodine or tetracaine increased global intracellular Ca(2+) and constricted the arteries. The blockade of IP?Rs with xestospongin D (5 μM) or 2-aminoethoxydiphenyl borate (100 μM) or the inhibition of phospholipase C using U-73122 (10 μM) also attenuated Ca(2+) waves without affecting Ca(2+) sparks. Importantly, the IP?Rs and phospholipase C antagonists decreased global intracellular Ca(2+) and dilated the arteries. In contrast, cremaster arterioles displayed only Ca(2+) waves: Ca(2+) sparks were not observed, and neither ryanodine (10-50 μM) nor tetracaine (100 μM) affected either Ca(2+) signals or arteriolar tone despite the presence of functional RyRs as assessed by responses to the RyR agonist caffeine (10 mM). As in feed arteries, arteriolar Ca(2+) waves were attenuated by xestospongin D (5 μM), 2-aminoethoxydiphenyl borate (100 μM), and U-73122 (10 μM), accompanied by decreased global intracellular Ca(2+) and vasodilation. These findings highlight the contrasting roles played by RyRs and IP?Rs in Ca(2+) signals and myogenic tone in feed arteries and demonstrate important differences in the function of RyRs between feed arteries and downstream arterioles.     

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).     

Structure and function of tyrosine kinase receptors

Over the past ten years, several growth factor receptors have been shown to be ligand-regulated tyrosine kinases. Tyrosine kinase activity is essential for signal transmission, suggesting that phosphorylation cascades may play an important role. Considerable effort has gone into understanding the structure and function of tyrosine kinase receptors in order to define their mechanisms of signal transmission. However, the protein substrates of the receptor kinases have proven to be difficult to isolate and clone. This review focuses on the receptors for insulin, epidermal growth factor, and platelet-derived growth factor. They are all tyrosine kinases, but emerging evidence suggests that they utilize multiple separate signal transduction pathways. Work carried out during the next several years should yield considerable insight into the complexity of the components which interact with these tyrosine kinase receptors to regulate cellular growth and metabolism.     

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.     

Structure and function of A1 adenosine receptors

The A1 adenosine receptor is the best characterized of the widely distributed purinergic receptor family. The purified brain A1 receptor is a monomeric 35- to 36-kDa glycoprotein. A1 receptors can be clearly distinguished from A2 adenosine receptors on the basis of structure activity relationships with selective ligands. Recent structure activity data suggest that subtypes of A1 (A1a, A1b, and A3) and A2 (A2a and A2b) receptors may exist. A1 receptor-mediated responses are coupled via multiple pertussis toxin-sensitive GTP binding proteins (G proteins) to many different effectors in various tissues: adenylate cyclase, phospholipase C, Na+- Ca2+ exchange, Ca2+ channels, Cl- channels, and K+ channels. The formation of calcium-mobilizing inositol phosphates can either be enhanced or inhibited. In general, adenosine has been found to act in concert with other hormones or neurotransmitters in either an inhibitory or a stimulatory way. The myriad modulatory actions of adenosine suggest that: 1) adenosine may simultaneously produce multiple effects within the same cell; and 2) activation of A1 receptors may lead to either a decrease or an increase in the coupling of other receptors to their G proteins.     

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.