Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.     

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.     

Mitochondria efficiently buffer subplasmalemmal Ca2+ elevation during agonist stimulation

In endothelial cells, local Ca(2+) release from superficial endoplasmic reticulum (ER) activates BK(Ca) channels. The resulting hyperpolarization promotes capacitative Ca(2+) entry (CCE), which, unlike BK(Ca) channels, is inhibited by high Ca(2+). To understand how the coordinated activation of plasma membrane ion channels with opposite Ca(2+) sensitivity is orchestrated, the individual contribution of mitochondria and ER in regulation of subplasmalemmal Ca(2+) concentration ([Ca(2+)](pm)) was investigated. For organelle visualization, cells were transfected with DsRed and yellow cameleon targeted to mitochondria and ER. The patch pipette was placed far from any organelle (L1), close to ER (L3), or mitochondria (L2) and activity of BK(Ca) channels was used to estimate local [Ca(2+)](pm). Under standard patch conditions (130 mm K(+) in the bath), histamine increased [Ca(2+)](pm) at L1 and L3 to approximately 1.6 microm, whereas close to mitochondria [Ca(2+)](pm) remained unchanged. If mitochondria moved apart from the pipette or in the presence of carbonyl cyanide-4-trifluoromethoxyphenylhyrazone, [Ca(2+)](pm) at L2 increased in response to histamine. Under standard patch conditions Ca(2+) entry was negligible due to cell depolarization. Using a physiological patch approach (5.6 mm K(+) in the bath), changes in [Ca(2+)](pm) to histamine could be monitored without cell depolarization and, thus, in conditions where Ca(2+) entry occurred. Here, histamine induced an initial transient Ca(2+) elevation to > or =3.5 microm followed by a long lasting plateau at approximately 1.2 microm in L1 and L3, whereas mitochondria kept neighboring [Ca(2+)](pm) low during stimulation. Thus, superficial mitochondria and ER generate local domains of low and high Ca(2+) allowing simultaneous activation of BK(Ca) and CCE, despite their opposite Ca(2+) sensitivity.     

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.     

Reduced threshold for luminal Ca2+ activation of RyR1 underlies a causal mechanism of porcine malignant hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.     

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.     

Functional defects in six ryanodine receptor isoform-1 (RyR1) mutations associated with malignant hyperthermia and their impact on skeletal excitation-contraction coupling

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder of skeletal muscle that segregates with >60 mutations within the MHS-1 locus on chromosome 19 coding for ryanodine receptor type 1 (RyR1). Although some MHRyR1s have been shown to enhance sensitivity to caffeine and halothane when expressed in non-muscle cells, their influence on EC coupling can only be studied in skeletal myotubes. We therefore expressed WTRyR1, six of the most common human MHRyR1s (R163C, G341R, R614C, R2163C, V2168M, and R2458H), and a newly identified C-terminal mutation (T4826I) in dyspedic myotubes to study their functional defects and how they influence EC coupling. Myotubes expressing any MHRyR1 were significantly more sensitive to stimulation by caffeine and 4-CmC than those expressing WTRyR1. The hypersensitivity of MH myotubes extended to K+ depolarization. MH myotubes responded to direct channel activators with maximum Ca2+ amplitudes consistently smaller than WT myotubes, whereas the amplitude of their responses to depolarization were consistently larger than WT myotubes. The magnitudes of responses attainable from myotubes expressing MHRyR1s are therefore related to the nature of the stimulus rather than size of the Ca2+ store. The functional changes of MHRyR1s were directly analyzed using [3H]ryanodine binding analysis of isolated myotube membranes. Although none of the MHRyR1s examined significantly altered EC50 for Ca2+ activation, many failed to be completely inhibited by a low Ca2+ (相似文献   

Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human coagulation factor xa. Studies on catalytic efficiency and inhibitor binding

The serine protease domain of factor Xa (FXa) contains a sodium as well as a calcium-binding site. Here, we investigated the functional significance of these two cation-binding sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na(+) binds to the substrate bound FXa with K(d) approximately 39 mm in the absence and approximately 9.5 mm in the presence of Ca(2+). Sodium-bound FXa (sodium-Xa) has approximately 18-fold increased catalytic efficiency ( approximately 4.5-fold decrease in K(m) and approximately 4-fold increase in k(cat)) in hydrolyzing S-2222 (benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide), and Ca(2+) further increases this k(cat) approximately 1.4-fold. Ca(2+) binds to the protease domain of substrate bound FXa with K(d) approximately 705 microm in the absence and approximately 175 microm in the presence of Na(+). Ca(2+) binding to the protease domain of FXa (Xa-calcium) has no effect on the K(m) but increases the k(cat) approximately 4-fold in hydrolyzing S-2222, and Na(+) further increases this k(cat) approximately 1.4-fold. In agreement with the K(m) data, sodium-Xa has approximately 5-fold increased affinity in its interaction with p-aminobenzamidine (S1 site probe) and approximately 4-fold increased rate in binding to the two-domain tissue factor pathway inhibitor; Ca(2+) (+/-Na(+)) has no effect on these interactions. Antithrombin binds to Xa-calcium with a approximately 4-fold faster rate, to sodium-Xa with a approximately 24-fold faster rate and to sodium-Xa-calcium with a approximately 28-fold faster rate. Thus, Ca(2+) and Na(+) together increase the catalytic efficiency of FXa approximately 28-fold. Na(+) enhances Ca(2+) binding, and Ca(2+) enhances Na(+) binding. Further, Na(+) enhances S1 site occupancy, and S1 site occupancy enhances Na(+) binding. Therefore, Na(+) site is thermodynamically linked to the S1 site as well as to the protease domain Ca(2+) site, whereas Ca(2+) site is only linked to the Na(+) site. The significance of these findings is that during physiologic coagulation, most of the FXa formed will exist as sodium-Xa-calcium, which has maximum biologic activity.     

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.     

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.     

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+).     

Influence of external K+ on potassium efflux in isolated chicken enterocytes.

1. Efflux of K+ was measured in pre-loaded (86Rb+) chicken enterocytes incubated in buffers with external K+ concentration ([K+]0) between 1 and 40 mM. 2. A decrease in [K+]0 from 6 to 1 mM reduced the rate constant of K+ efflux, whereas it was stimulated by increasing [K+]0 from 6 to 40 mM. 3. The inhibitory effect of low [K+]0 on K+ efflux was: (i) higher than that expected from a change in the electrical driving force, suggesting that membrane K+ permeability has been decreased, and (ii) attenuated by A23187 and Na(+)-free buffers. 4. The effect of A23187 on K(+)-induced K+ efflux was abolished by apamin and that of Na(+)-free buffers by apamin, quinine or verapamil, which suggests that the effect of low K+ on K+ efflux seems to be due to decreased intracellular Ca2+ concentration. 5. The stimulatory effect of 40 mM K0+ on K+ exit can be accounted for by an increase in the electrical driving force. 6. The efflux of K+ at 40 mM K0 appears to occur through Ca2(+)-activated K+ channels (KCa) since it was prevented by 500 microM quinine and unaffected by bumetanide or 3,4-diaminopyridine. 7. In addition, the current results show that an increase in external K+ concentration reduced the ability of quinine to inhibit KCa channels, and even abolished that of Ba2+ and apamin.     

Electrophysiological effects of orexins/hypocretins on pedunculopontine tegmental neurons in rats: an in vitro study

Orexin-A (ORX-A) and orexin-B (ORX-B) play critical roles in the regulation of sleep-wakefulness and feeding. ORX neurons project to the pedunculopontine tegmental nucleus (PPT), which regulates waking and rapid eye movement (REM) sleep. Thus, we examined electrophysiological effects of ORXs on rat PPT neurons with a soma size of more than 30 microm. Whole cell patch clamp recording in vitro revealed that ORX-A and ORX-B depolarized PPT neurons dose-dependently in normal and/or tetrodotoxin containing artificial cerebrospinal fluids (ACSFs), and the EC(50) values for ORX-A and ORX-B were 66 nM and 536 nM, respectively. SB-334867, a selective inhibitor for ORX 1 (OX(1)) receptors, significantly suppressed the ORX-A-induced depolarization. The ORX-A-induced depolarization was reduced in high K(+) ACSF with extracellular K(+) concentration of 13.25 mM or N-methyl-d-glucamine (NMDG(+))-containing ACSF in which NaCl was replaced with NMDG-Cl, and abolished in high K(+)-NMDG(+) ACSF or in a combination of NMDG(+) ACSF and recordings with Cs(+)-containing pipettes. An inhibitor of Na(+)/Ca(2+) exchanger and chelating intracellular Ca(2+) had no effect on the depolarization. Most of PPT neurons studied were characterized by an A-current or both A-current and a low threshold Ca(2+) spike, and predominantly cholinergic. These results suggest that ORXs directly depolarize PPT neurons via OX(1) receptors and via a dual ionic mechanism including a decrease of K(+) conductances and an increase of non-selective cationic conductances, and support the notion that ORX neurons affect the activity of PPT neurons directly and/or indirectly to control sleep-wakefulness, especially REM sleep.     

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.     

Distinct effects on Ca2+ handling caused by malignant hyperthermia and central core disease mutations in RyR1

Malignant hyperthermia (MH) and central core disease (CCD) are disorders of skeletal muscle Ca2+ homeostasis that are linked to mutations in the type 1 ryanodine receptor (RyR1). Certain RyR1 mutations result in an MH-selective phenotype (MH-only), whereas others result in a mixed phenotype (MH + CCD). We characterized effects on Ca2+ handling and excitation-contraction (EC) coupling of MH-only and MH + CCD mutations in RyR1 after expression in skeletal myotubes derived from RyR1-null (dyspedic) mice. Compared to wild-type RyR1-expressing myotubes, MH + CCD- and MH-only-expressing myotubes exhibited voltage-gated Ca2+ release (VGCR) that activated at more negative potentials and displayed a significantly higher incidence of spontaneous Ca2+ oscillations. However, maximal VGCR was reduced only for MH + CCD mutants (Y4795C, R2435L, and R2163H) in which spontaneous Ca2+ oscillations occurred with significantly longer duration (Y4795C and R2435L) or higher frequency (R2163H). Notably, myotubes expressing these MH + CCD mutations in RyR1 exhibited both increased [Ca2+]i and reduced sarcoplasmic reticulum (SR) Ca2+ content. We conclude that MH-only mutations modestly increase basal release-channel activity in a manner insufficient to alter net SR Ca2+ content ("compensated leak"), whereas the mixed MH + CCD phenotype arises from mutations that enhance basal activity to a level sufficient to promote SR Ca2+ depletion, elevate [Ca2+]i, and reduce maximal VGCR ("decompensated leak").     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.     

Ca2+ signaling in microdomains: Homer1 mediates the interaction between RyR2 and Cav1.2 to regulate excitation-contraction coupling

Excitation-contraction (E-C) coupling and Ca(2+)-induced Ca(2+) release in smooth and cardiac muscles is mediated by the L-type Ca(2+) channel isoform Ca(v)1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca(v)1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca(v)1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1(-/-) mice and by demonstrating a Homer1-mediated dynamic interaction between Ca(v)1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca(v)1.2. Most notably, expression of Ca(v)1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca(v)1.2 and RyR2 to regulate the intensity of Ca(2+)-induced Ca(2+) release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca(v)1.2 and RyR2. In one state, Ca(v)1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca(v)1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca(v)1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.     

Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K(+) channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K(+) and Ca(2+) conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.