Skeletal and cardiac ryanodine receptors exhibit different responses to Ca2+ overload and luminal ca2+

Spontaneous Ca(2+) release occurs in cardiac cells during sarcoplasmic reticulum Ca(2+) overload, a process we refer to as store-overload-induced Ca(2+) release (SOICR). Unlike cardiac cells, skeletal muscle cells exhibit little SOICR activity. The molecular basis of this difference is not well defined. In this study, we investigated the SOICR properties of HEK293 cells expressing RyR1 or RyR2. We found that HEK293 cells expressing RyR2 exhibited robust SOICR activity, whereas no SOICR activity was observed in HEK293 cells expressing RyR1. However, in the presence of low concentrations of caffeine, SOICR could be triggered in these RyR1-expressing cells. At the single-channel level, we showed that RyR2 is much more sensitive to luminal Ca(2+) than RyR1. To identify the molecular determinants responsible for these differences, we constructed two chimeras between RyR1 and RyR2, N-RyR1(1-4006)/C-RyR2(3962-4968) and N-RyR2(1-3961)/C-RyR1(4007-5037). We found that replacing the C-terminal region of RyR1 with the corresponding region of RyR2 (N-RyR1/C-RyR2) dramatically enhanced the propensity for SOICR and the response to luminal Ca(2+), whereas replacing the C-terminal region of RyR2 with the corresponding region of RyR1 (N-RyR2/C-RyR1) reduced the propensity for SOICR and the luminal Ca(2+) response. These observations indicate that the C-terminal region of RyR is a critical determinant of both SOICR and the response to luminal Ca(2+). These chimeric studies also reveal that the N-terminal region of RyR plays an important role in regulating SOICR and luminal Ca(2+) response. Taken together, our results demonstrate that RyR1 differs markedly from RyR2 with respect to their responses to Ca(2+) overload and luminal Ca(2+), and suggest that the lack of spontaneous Ca(2+) release in skeletal muscle cells is, in part, attributable to the unique intrinsic properties of RyR1.     

Beyond Bowditch: the convergence of cardiac chronotropy and inotropy

The ability of the heart to acutely beat faster and stronger is central to the vertebrate survival instinct. Released neurotransmitters, norepinephrine and epinephrine, bind to beta-adrenergic receptors (beta-AR) on pacemaker cells comprising the sinoatrial node, and to beta-AR on ventricular myocytes to modulate cellular mechanisms that govern the frequency and amplitude, respectively, of the duty cycles of these cells. While a role for sarcoplasmic reticulum Ca(2+) cycling via SERCA2 and ryanodine receptors (RyR) has long been appreciated with respect to cardiac inotropy, recent evidence also implicates Ca(2+) cycling with respect to chronotropy. In spontaneously beating primary sinoatrial nodal pacemaker cells, RyR Ca(2+) releases occurring during diastolic depolarization activate the Na(+)-Ca(2+) exchanger (NCX) to produce an inward current that enhances their diastolic depolarization rate, and thus increases their beating rate. beta-AR stimulation synchronizes RyR activation and Ca(2+) release to effect an increased beating rate in pacemaker cells and contraction amplitude in myocytes: in pacemaker cells, the beta-AR stimulation synchronization of RyR activation occurs during the diastolic depolarization, and augments the NCX inward current; in ventricular myocytes, beta-AR stimulation synchronizes the openings of unitary L-type Ca(2+) channel activation following the action potential, and also synchronizes RyR Ca(2+) releases following depolarization, and in the absence of depolarization, both leading to the generation of a global cytosolic Ca(i) transient of increased amplitude and accelerated kinetics. Thus, beta-AR stimulation induced synchronization of RyR activation (recruitment of additional RyRs to fire) and of the ensuing Ca(2+) release cause the heart to beat both stronger and faster, and is thus, a common mechanism that links both the maximum achievable cardiac inotropy and chronotropy.     

Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.     

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.     

Maltooligosaccharide disproportionation reaction: an intrinsic property of amylosucrase from Neisseria polysaccharea

Ca(2+) signaling plays an important role in the function of dendritic cells (DC), the specialized antigen-presenting cells of the immune system. Here we describe functional ryanodine receptor (RyR) Ca(2+) release channels in murine, bone marrow-derived DC. RT-PCR analysis identified selective expression of the type 1 RyR, with higher levels detected in immature rather than mature DC. The RyR activators caffeine, FK506, ryanodine and 4-chloro-m-cresol mobilized Ca(2+) in DC, and responses to 4-chloro-m-cresol were inhibited by dantrolene. Furthermore, activation of RyRs both inhibited subsequent inositol trisphosphate-mediated Ca(2+) release and provoked store-operated Ca(2+) entry, suggesting a functional interaction between these intracellular Ca(2+) channels. Thus, the RyR1 channel may play an intrinsic role in Ca(2+) signaling in DC.     

Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling.

In excitable cells membrane depolarization is translated into intracellular Ca2+ signals. The ryanodine receptor (RyR) amplifies the Ca2+ signal by releasing Ca2+ from the intracellular Ca2+ store upon receipt of a message from the dihydropyridine receptor (DHPR) on the plasma membrane in striated muscle. There are two distinct mechanisms for the amplification of Ca2+ signalling. In cardiac cells depolarization-dependent Ca2+ influx through DHPR triggers Ca2+-induced Ca2+ release via RyR, while in skeletal muscle cells a voltage-induced change in DHPR is thought to be mechanically transmitted, without a requirement for Ca2+ influx, to RyR to cause it to open. In expression experiments using mutant skeletal myocytes lacking an intrinsic subtype of RyR (RyR-1), we demonstrate that RyR-1, but not the cardiac subtype (RyR-2), is capable of supporting skeletal muscle-type coupling. Furthermore, when RyR-2 was expressed in skeletal myocytes, we observed depolarization-independent spontaneous Ca2+ waves and oscillations, which suggests that RyR-2 is prone to regenerative Ca2+ release responses. These results demonstrate functional diversity among RyR subtypes and indicate that the subtype of RyR is the key to Ca2+ signal amplification.     

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Regulation of ryanodine receptor opening by lumenal Ca(2+) underlies quantal Ca(2+) release in PC12 cells

Graded or "quantal" Ca(2+) release from intracellular stores has been observed in various cell types following activation of either ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (InsP(3)R). The mechanism causing the release of Ca(2+) stores in direct proportion to the strength of stimulation is unresolved. We investigated the properties of quantal Ca(2+) release evoked by activation of RyR in PC12 cells, and in particular whether the sensitivity of RyR to the agonist caffeine was altered by lumenal Ca(2+). Quantal Ca(2+) release was observed in cells stimulated with 1 to 40 mM caffeine, a range of caffeine concentrations giving a >10-fold change in lumenal Ca(2+) content. The Ca(2+) load of the caffeine-sensitive stores was modulated by allowing them to refill for varying times after complete discharge with maximal caffeine, or by depolarizing the cells with K(+) to enhance their normal steady-state loading. The threshold for RyR activation was sensitized approximately 10-fold as the Ca(2+) load increased from a minimal to a maximal loading. In addition, the fraction of Ca(2+) released by low caffeine concentrations increased. Our data suggest that RyR are sensitive to lumenal Ca(2+) over the full range of Ca(2+) loads that can be achieved in an intact PC12 cell, and that changes in RyR sensitivity may be responsible for the termination of Ca(2+) release underlying the quantal effect.     

FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 μM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias.     

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).     

ATP-induced activation of expressed RyR3 at low free calcium

Ca(2+) channel properties of the mink ryanodine receptor type 3 (RyR3), expressed in HEK293 cells, were studied in planar lipid bilayers to which RyR3 rich membrane fragments had been fused. RyR3 channels were not active at resting levels of Ca(2+)(free) but were gated by an additional 1 mM ATP, exhibiting long open times. The second major finding was the absence of channel inactivation at millimolar Ca(2+)(free). Insertion of a myc tag at the N-terminus of RyR3 did not affect the channel properties. As to skeletal muscle, the observed type 3 channel properties appear physiologically meaningful by assisting type 1 channels in calcium release.     

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.     

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.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle.

The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.     

The cytosolic N-terminus of presenilin-1 potentiates mouse ryanodine receptor single channel activity

Ryanodine receptors (RyRs) amplify intracellular Ca(2+) signals by massively releasing Ca(2+) from intracellular stores. Exaggerated chronic Ca(2+) release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca(2+)-dependent apoptosis, providing a basis for the "calcium hypothesis" of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF(1-82)), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF(1-82) strongly increased both mean currents (EC(50)=12nM, Hill coefficient (n(H)) approximately 1) and open probability for higher sublevels for single RyR channels (EC(50)=7nM, n(H) approximately 2). Bell-shaped Ca(2+)-activation curve remained unchanged, suggesting that PS1 NTF(1-82) allosterically potentiates RyRs, but that the channel still requires Ca(2+) for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF(1-82) was overridden by receptor desensitization at high [Ca(2+)] (pCa>5). This potentiation of RyR by PS1 NTF(1-82) reveals a new mechanism of physiologically relevant PS1-regulated Ca(2+) release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca(2+) leak channels formed by PS1 holoproteins.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.