Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Lack of effect of cADP-ribose and NAADP on the activity of skeletal muscle and heart ryanodine receptors

The calcium release channels/ryanodine receptors (RyRs) are potential/putative targets of cADPR (cyclic ADP-ribose) action in many tissue systems. In striated muscles, where RyRs predominate, cADPR action on these channels is controversial. Here cADPR modulation of cardiac and skeletal muscle RyR channels was tested. We considered factors reported as necessary for cADPR action, such as the presence of calmodulin and/or FK binding proteins (FKBPs). We found: 1) The RyR channel isoforms were insensitive to cADPR (or its metabolite NAADP [nicotinic acid adenine dinucleotide phosphate]) under all conditions examined, as studied by: 1a) single channel recordings in planar lipid bilayers; 1b) macroscopic behavior of the RyRs in sarcoplasmic reticulum (SR) microsomes (including crude microsome preparations likely to retain putative cADPR cofactors) at room temperature and at 37 degrees C (net energized Ca2+ uptake or passive Ca2+ leak); 2) [32P]cADPR did not bind significantly to SR microsomes; 3) cADPR did not affect FKBP association to SR membranes. We conclude that cADPR does not interact directly with RyRs or RyR-associated SR proteins. Our results under in vitro conditions suggest that c ADPR effects on Ca2+ signaling observed in vivo in mammalian striated muscle cells may reflect indirect modulation of RyRs or RyR-independent Ca2+ release systems.     

Role of FKBP12.6 in cADPR-induced activation of reconstituted ryanodine receptors from arterial smooth muscle

cADP ribose (cADPR) serves as second messenger to activate the ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) and mobilize intracellular Ca(2+) in vascular smooth muscle cells. However, the mechanisms mediating the effect of cADPR remain unknown. The present study was designed to determine whether FK-506 binding protein 12.6 (FKBP12.6), an accessory protein of the RyRs, plays a role in cADPR-induced activation of the RyRs. A 12.6-kDa protein was detected in bovine coronary arterial smooth muscle (BCASM) and cultured CASM cells by being immunoblotted with an antibody against FKBP12, which also reacted with FKBP12.6. With the use of planar lipid bilayer clamping techniques, FK-506 (0.01-10 microM) significantly increased the open probability (NP(O)) of reconstituted RyR/Ca(2+) release channels from the SR of CASM. This FK-506-induced activation of RyR/Ca(2+) release channels was abolished by pretreatment with anti-FKBP12 antibody. The RyRs activator cADPR (0.1-10 microM) markedly increased the activity of RyR/Ca(2+) release channels. In the presence of FK-506, cADPR did not further increase the NP(O) of RyR/Ca(2+) release channels. Addition of anti-FKBP12 antibody also completely blocked cADPR-induced activation of these channels, and removal of FKBP12.6 by preincubation with FK-506 and subsequent gradient centrifugation abolished cADPR-induced increase in the NP(O) of RyR/Ca(2+) release channels. We conclude that FKBP12.6 plays a critical role in mediating cADPR-induced activation of RyR/Ca(2+) release channels from the SR of BCASM.     

The CD38-cyclic ADP-ribose signaling system in insulin secretion

Glucose induces an increase in the intracellular Ca2+ concentration in pancreatic -cells to secrete insulin. CD38 occurs in -cells and has both ADP-ribosyl cyclase, which catalyzes the formation of cyclic ADP-ribose (cADPR) from NAD+, and cADPR hydrolase, which converts cADPR to ADP-ribose. ATP, produced by glucose metabolism, competes with cADPR for the binding site, Lys-129, of CD38, resulting in the inhibition of the hydrolysis of cADPR and thereby causing cADPR accumulation in -cells. Cyclic ADP-ribose then binds to FK506-binding protein 12.6 in the ryanodine receptor Ca2+ channel (RyR), dissociating the binding protein from RyR to induce the release of Ca2+ from the endoplasmic reticulum. Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) phosphorylates RyR to sensitize and activate the Ca2+ channel. Ca2+, released from the RyR, further activates CaM kinase II and amplifies the process. Thus, cADPR acts as a second messenger for Ca2+ mobilization to secrete insulin. The novel mechanism of insulin secretion described above is different from the conventional hypothesis in which Ca2+ influx from extracellular sources plays a role in insulin secretion by glucose.     

Selected contribution: effect of volatile anesthetics on cADP-ribose-induced Ca(2+) release system.

Volatile anesthetics have multiple actions on intracellular Ca(2+) homeostasis, including activation of the ryanodine channel (RyR) and sensitization of this channel to agonists such as caffeine and ryanodine. Recently it has been described that the nucleotide cADP-ribose (cADPR) is the endogenous regulator of the RyR in many mammalian cells, and cADPR has been proposed to be a second messenger in many signaling pathways. I investigated the effect of volatile anesthetics on the cADPR signaling system, using sea urchin egg homogenates as a model of intracellular Ca(2+) stores. Ca(2+) uptake and release were monitored in sea urchin egg homogenates by using the fluo-3 fluorescence technique. Activity of the ADP-ribosyl cyclase was monitored by using a fluorometric method using nicotinamide guanine dinucleotide as a substrate. Halothane in concentrations up to 800 microM did not induce Ca(2+) release by itself in sea urchin egg homogenates. However, halothane potentiates the Ca(2+) release mediated by agonists of the ryanodine channel, such as ryanodine. Furthermore, other volatile anesthetics such as isoflurane and sevoflurane had no effect. Halothane also potentiated the activation of the ryanodine channel mediated by the endogenous nucleotide cADPR. The half-maximal concentration for cADPR-induced Ca(2+) release was decreased about three times by addition of 800 microM halothane. The reverse was also true: addition of subthreshold concentrations of cADPR sensitized the homogenates to halothane. In contrast, all the volatile anesthetics used had no effect on the activity of the enzyme that synthesizes cADPR. I propose that the complex effect of volatile anesthetics on intracellular Ca(2+) homeostasis may involve modulation of the cADPR signaling system.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Interactions between calcium release pathways: multiple messengers and multiple stores

The discovery of cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca(2+) releasing messengers has provided additional insight into how complex Ca(2+) signalling patterns are generated. There is mounting evidence that these molecules along with the more established messenger, myo-inositol 1,4,5-trisphosphate (IP(3)), have a widespread messenger role in shaping Ca(2+) signals in many cell types. These molecules have distinct structures and act on specific Ca(2+) release mechanisms. Emerging principles are that cADPR enhances the Ca(2+) sensitivity of ryanodine receptors (RYRs) to produce prolonged Ca(2+) signals through Ca(2+)-induced Ca(2+) release (CICR), while NAADP acts on a novel Ca(2+) release mechanism to produce a local trigger Ca(2+) signal which can be amplified by CICR by recruiting other Ca(2+) release mechanisms. Whilst IP(3) and cADPR mobilise Ca(2+) from the endoplasmic reticulum (ER), recent evidence from the sea urchin egg suggests that the major NAADP-sensitive Ca(2+) stores are reserve granules, acidic lysosomal-related organelles.In this review we summarise the role of multiple Ca(2+) mobilising messengers, Ca(2+) release channels and Ca(2+) stores, and the interplay between them, in the generation of specific Ca(2+) signals. Focusing upon cADPR and NAADP, we discuss how cellular stimuli may draw upon different combinations of these messengers to produce distinct Ca(2+) signalling signatures.     

A calmodulin binding domain of RyR increases activation of spontaneous Ca2+ sparks in frog skeletal muscle

The calmodulin C lobe binding region (residues 3614-3643) on the sarcoplasmic reticulum Ca2+ release channel (RyR1) is thought to be a region of contact between subunits within RyR1 homotetramer Ca2+ release channels. To determine whether the 3614-3643 region is a regulatory site/interaction domain within RyR in muscle fibers, we have investigated the effect of a synthetic peptide corresponding to this region (R3614-3643) on Ca2+ sparks in frog skeletal muscle fibers. R3614-3643 (0.2-3.0 microM) promoted the occurrence of Ca2+ sparks in a highly cooperative dose-dependent manner, with a half-maximal activation at 0.47 microM and a maximal increase in frequency of approximately 5-fold. A peptide with a single amino acid substitution within R3614-3643 (L3624D) retained the ability to bind Ca(2+)-free calmodulin but did not increase Ca2+ spark frequency, suggesting that R3614-3643 does not modulate Ca2+ sparks by removal of endogenous calmodulin. Our data support a model in which the calmodulin binding domain of RyR1 modulates channel activity by at least two mechanisms: direct binding of calmodulin as well as interactions with other regions of RyR.     

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.     

Ca2+ release induced by cyclic ADP ribose in mice lacking type 3 ryanodine receptor.

The action of cyclic-ADP-ribose was studied on calcium release from sarcoplasmic reticulum of skeletal muscles of neonatal and adult wild-type and RyR3-deficient mice. cADPR increased calcium efflux from microsomes, enhanced caffeine-induced calcium release, and, in 20% of the tests, triggered calcium release in single muscle fibers. These responses occurred only in the diaphragm of adult RyR3-deficient mice. cADPR action was abolished by ryanodine, ruthenium red, and 8-brome-cADPR. These results strongly favor a specific action of cADPR on RyR1. The responsiveness of RyR1 appears in adult muscles when RyR3 is lacking.     

Depletion of intracellular Ca2+ by caffeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor

Recent studies have suggested a central role for Ca(2+) in the signaling pathway of apoptosis and certain anti-apoptotic effects of Bcl-2 family of proteins have been attributed to changes in intracellular Ca(2+) homeostasis. Here we report that depletion of Ca(2+) from endoplasmic reticulum (ER) leads to apoptosis in Chinese hamster ovary cells. Stable expression of ryanodine receptor (RyR) in these cells enables rapid and reversible changes of both cytosolic Ca(2+) and ER Ca(2+) content via activation of the RyR/Ca(2+) release channel by caffeine and ryanodine. Sustained depletion of the ER Ca(2+) store leads to apoptosis in Chinese hamster ovary cells, whereas co-expression of Bcl-xL and RyR in these cells prevents apoptotic cell death but not necrotic cell death. The anti-apoptotic effect of Bcl-xL does not correlate with changes in either the Ca(2+) release process from the ER or the capacitative Ca(2+) entry through the plasma membrane. The data suggest that Bcl-xL likely prevents apoptosis of cells at a stage downstream of ER Ca(2+) release and capacitative Ca(2+) entry.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

RyR3 amplifies RyR1-mediated Ca(2+)-induced Ca(2+) release in neonatal mammalian skeletal muscle

The neonatal mammalian skeletal muscle contains both type 1 and type 3 ryanodine receptors (RyR1 and RyR3) located in the sarcoplasmic reticulum membrane. An allosteric interaction between RyR1 and dihydropyridine receptors located in the plasma membrane mediates voltage-induced Ca(2+) release (VICR) from the sarcoplasmic reticulum. RyR3, which disappears in adult muscle, is not involved in VICR, and the role of the transiently expressed RyR3 remains elusive. Here we demonstrate that RyR1 participates in both VICR and Ca(2+)-induced Ca(2+) release (CICR) and that RyR3 amplifies RyR1-mediated CICR in neonatal skeletal muscle. Confocal measurements of intracellular Ca(2+) in primary cultured mouse skeletal myotubes reveal active sites of Ca(2+) release caused by peripheral coupling between dihydropyridine receptors and RyR1. In myotubes lacking RyR3, the peripheral VICR component is unaffected, and RyR1s alone are able to support inward CICR propagation in most cells at an average speed of approximately 190 microm/s. With the co-presence of RyR1 and RyR3 in wild-type cells, unmitigated radial CICR propagates at 2,440 microm/s. Because neonatal skeletal muscle lacks a well developed transverse tubule system, the RyR3 reinforcement of CICR seems to ensure a robust, uniform, and synchronous activation of Ca(2+) release throughout the cell body. Such functional interplay between RyR1 and RyR3 can serve important roles in Ca(2+) signaling of cell differentiation and muscle contraction.     

Mechanism of calmodulin inhibition of cardiac sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor)

The functional effects of calmodulin (CaM) on single cardiac sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors) (RyR2s) were determined in the presence of two endogenous channel effectors, MgATP and reduced glutathione, using the planar lipid bilayer method. Single-channel activities, number of events, and open and close times were determined at varying cytosolic Ca(2+) concentrations. CaM reduced channel open probability at <10 micro M Ca(2+) by decreasing channel events and mean open times and increasing mean close times. At >10 micro M Ca(2+), CaM was less effective in inhibiting RyR2. CaM decreased mean open times but increased channel events, without significantly affecting mean close times. A series of voltage pulses was applied to the bilayer from +50 to -50 mV and from -50 mV to +50 mV to rapidly increase and decrease open channel-mediated sarcoplasmic reticulum lumenal to cytosolic Ca(2+) fluxes. CaM decreased the duration of the open events after the voltage switch from -50 mV to +50 mV. In parallel experiments, a Ca(2+)-insensitive calmodulin mutant was without effect on RyR2 activity. The results are discussed in terms of a possible role of CaM in the termination of cardiac sarcoplasmic reticulum Ca(2+) release.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

Microinjection of cyclic ADP-ribose triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in medaka eggs

Medaka (Oryzias latipes) eggs microinjected with the Ca(2+)-mobilising messenger cyclic adenosine diphosphate ribose (cADPR) underwent a wave of exocytosis of cortical alveoli and were thus activated. The number of eggs activated was sharply dependent on the concentration of cADPR in the pipette, the threshold concentration was approximately 60 nM. After injection, a pronounced latency preceded the onset of cortical alveoli exocytosis; this latency was independent of the concentration of cADPR but decreased markedly with increasing temperature. Heat-treated cADPR, which yields the inert non-cyclised product ADP-ribose, was ineffective in activating eggs. When cADPR was injected into aequorin-loaded eggs, a wave of luminescence arose at the site of cADPR injection and then swept out across the egg with a mean velocity of approximately 13 microns/s; the velocity was independent of the concentration of injected cADPR. In such a large cell (diameter of around 1 mm), this is considerably faster than that possible by simple diffusion of cADPR, which unambiguously demonstrates that cADPR must activate a regenerative process. cADPR has been demonstrated to modulate Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors (RyRs) in many cell types, and consistent with this was the finding that microinjection of the pharmacological RyR modulator, ryanodine, also activated medaka eggs. These results suggest that a cADPR-sensitive Ca2+ release mechanism is present in the medaka egg, that cADPR is the most potent activator of medaka eggs described to date, and that it activates eggs by triggering a wave of CICR from internal stores that in turn stimulates a wave of exocytosis.     

Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release

The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca(2+) release from intracellular Ca(2+) stores. How RyR's selectivity between the physiological cations K(+), Mg(2+), and Ca(2+) affects single-channel Ca(2+) current amplitude is examined using a recent model of RyR permeation. It is found that K(+) provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca(2+) release. Moreover, intra-pore competition between Ca(2+) and Mg(2+) defines single RyR Ca(2+) current amplitude. Since both [Mg(2+)] and [Ca(2+)](SR) can change during pathophysiological conditions, the RyR unitary Ca(2+) current amplitude during Ca(2+) release may change significantly due to this Ca(2+)/Mg(2+) competition. Compared to the classic action of Mg(2+) on RyR open probability, these Ca(2+) current amplitude changes have as large or larger effects on overall RyR Ca(2+) mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.     

Glucose and endoplasmic reticulum calcium channels regulate HIF-1beta via presenilin in pancreatic beta-cells

Pancreatic beta-cell death is a critical event in type 1 diabetes, type 2 diabetes, and clinical islet transplantation. We have previously shown that prolonged block of ryanodine receptor (RyR)-gated release from intracellular Ca(2+) stores activates calpain-10-dependent apoptosis in beta-cells. In the present study, we further characterized intracellular Ca(2+) channel expression and function in human islets and the MIN6 beta-cell line. All three RyR isoforms were identified in human islets and MIN6 cells, and these endoplasmic reticulum channels were observed in close proximity to mitochondria. Blocking RyR channels, but not sarco/endoplasmic reticulum ATPase (SERCA) pumps, reduced the ATP/ADP ratio. Blocking Ca(2+) flux through RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of hypoxia-inducible factor (HIF-1beta). Moreover, inhibition of RyR or inositol trisphosphate receptor channels, but not SERCA pumps, increased the expression of presenilin-1. Both HIF-1beta and presenilin-1 expression were also induced by low glucose. Overexpression of presenilin-1 increased HIF-1beta, suggesting that HIF is downstream of presenilin. Our results provide the first evidence of a presenilin-HIF signaling network in beta-cells. We demonstrate that this pathway is controlled by Ca(2+) flux through intracellular channels, likely via changes in mitochondrial metabolism and ATP. These findings provide a mechanistic understanding of the signaling pathways activated when intracellular Ca(2+) homeostasis and metabolic activity are suppressed in diabetes and islet transplantation.