Ligand-specific conformation of extracellular loop-2 in the angiotensin II type 1 receptor.

The orientation of the second extracellular loop (ECL2) is divergent in G-protein coupled receptor (GPCR) structures determined. This discovery provoked the question, is the ECL2 conformation differentially regulated in the GPCRs that respond to diffusible ligands? We have determined the conformation of the ECL2 of the angiotensin II type 1 receptor by reporter-cysteine accessibility mapping in different receptor states (i.e. empty, agonist-bound and antagonist-bound). We introduced cysteines at each position of ECL2 of an N-terminal epitope-tagged receptor surrogate lacking all non-essential cysteines and then measured reaction of these with a cysteine-reactive biotin probe. The ability of biotinylated mutant receptors to react with a steptavidin-HRP-conjugated antibody was used as the basis for examining differences in accessibility. Two segments of ECL2 were accessible in the empty receptor, indicating an open conformation of ECL2. These segments were inaccessible in the ligand-bound states of the receptor. Using the accessibility constraint, we performed molecular dynamics simulation to predict ECL2 conformation in different states of the receptor. Analysis suggested that a lid conformation similar to that of ECL2 in rhodopsin was induced upon binding both agonist and antagonist, but exposing different accessible segments delimited by the highly conserved disulfide bond. Our study reveals the ability of ECL2 to interact with diffusing ligands and to adopt a ligand-specific lid conformation, thus, slowing down dissociation of ligands when bound. Distinct conformations induced by the bound agonist and the antagonist around the conserved disulfide bond suggest an important role for this disulfide bond in producing different functional states of the receptor.

Signaling pathways from cannabinoid receptor-1 activation to inhibition of N-methyl-D-aspartic acid mediated calcium influx and neurotoxicity in dorsal root ganglion neurons.

Although the activation of cannabinoid receptor-1 (CB1) receptors by cannabinoids is known to inhibit neuronal hyperexcitability and reduce excitotoxic cell death, the mechanistic links between these two actions remain elusive. We tested the hypothesis that activation of CB1 receptors inhibits N-methyl-d-aspartic acid (NMDA)-mediated calcium influx and cell death via the inositol triphosphate (IP(3)) signaling pathway in both primary dorsal root ganglia neurons and a cultured neuronal cell line (F-11 cells). These cells were pretreated with the cannabinoid agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (R-(+)-WIN 55,212-2; WIN) before exposure to NMDA. Concentrations of cytosolic calcium were measured with the ratiometric calcium indicator, Fura-2, and cell death was determined by a cell viability test. WIN dose-dependently attenuated both the calcium influx and cell death induced by NMDA. These effects were blocked by selective cannabinoid CB1 receptor antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) or N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), but not CB2 receptor antagonist N-[(1S)-endo-1,3,3,-trimethylbicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide (SR144528). It is interesting to note that a transient Ca(2+) signal was observed after the acute application of WIN. This Ca(2+) increase was blocked by a CB1 receptor antagonist AM251, IP(3) receptor antagonist 2- aminoethyl diphenylborinate, or by depleting intracellular Ca(2+) stores with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin. Removal of extracellular Ca(2+), on the other hand, had no effect on the CB1 receptor-induced Ca(2+) increase. These data suggest that WIN triggers a cascade of events: it activates the CB1 receptor and the IP(3) signaling pathway, stimulates the release of Ca(2+) from intracellular stores, raises the cytosolic Ca(2+) levels, and inhibits the NMDA-mediated Ca(2+) influx and cell death through a process that remains to be determined.

Elucidation of a TRPC6-TRPC5 channel cascade that restricts endothelial cell movement.

Canonical transient receptor potential (TRPC) channels are opened by classical signal transduction events initiated by receptor activation or depletion of intracellular calcium stores. Here, we report a novel mechanism for opening TRPC channels in which TRPC6 activation initiates a cascade resulting in TRPC5 translocation. When endothelial cells (ECs) are incubated in lysophosphatidylcholine (lysoPC), rapid translocation of TRPC6 initiates calcium influx that results in externalization of TRPC5. Activation of this TRPC6-5 cascade causes a prolonged increase in intracellular calcium concentration ([Ca(2+)](i)) that inhibits EC movement. When TRPC5 is down-regulated with siRNA, the lysoPC-induced rise in [Ca(2+)](i) is shortened and the inhibition of EC migration is lessened. When TRPC6 is down-regulated or EC from TRPC6(-/-) mice are studied, lysoPC has minimal effect on [Ca(2+)](i) and EC migration. In addition, TRPC5 is not externalized in response to lysoPC, supporting the dependence of TRPC5 translocation on the opening of TRPC6 channels. Activation of this novel TRPC channel cascade by lysoPC, resulting in the inhibition of EC migration, could adversely impact on EC healing in atherosclerotic arteries where lysoPC is abundant.

The other side of the Maillard reaction.

The Maillard reaction plays an important role in eye lens aging and cataract formation. Methylglyoxal (MGO) is a metabolic dicarbonyl compound present in the lens. It reacts with arginine residues in lens proteins to form advanced glycation end products (AGEs), such as hydroimidazolones and argpyrimidine. alpha-Crystallin, comprising alphaA- and alphaB-crystallin, is a major protein of the lens and it functions as a chaperone protein. We have found that upon reaction with MGO, human alphaA-crystallin becomes a more effective chaperone. Modification of specific arginine residues to AGEs appears to be the reason. Mutation of these arginine residues to alanine mirrors the effect of MGO, suggesting neutralization of the positive charge on arginine residues as a cause for improved chaperone function. Reaction with MGO also blocks the loss of the chaperone function of alphaA-crystallin caused by nonenzymatic glycation by ascorbate and ribose. These findings suggest that low levels of MGO might help the lens remain transparent during aging.

Single-channel characterization of the rabbit recombinant RyR2 reveals a novel inactivation property of physiological concentrations of ATP.

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.

Manifold active-state conformations in GPCRs: agonist-activated constitutively active mutant AT1 receptor preferentially couples to Gq compared to the wild-type AT1 receptor.

The angiotensin II type I (AT(1)) receptor mediates regulation of blood pressure and water-electrolyte balance by Ang II. Substitution of Gly for Asn(111) of the AT(1) receptor constitutively activates the receptor leading to Gq-coupled IP(3) production independent of Ang II binding. The Ang II-activated conformation of the AT1(N111G) receptor was proposed to be similar to that of the wild-type AT(1) receptor, although, various aspects of the Ang II-induced conformation of this constitutively active mutant receptor have not been systematically studied. Here, we provide evidence that the conformation of the active state of the wild-type and the constitutively active AT(1) receptors are different. Upon Ang II binding an activated conformation of the wild-type AT(1) receptor activates G protein and recruits beta-arrestin. In contrast, the agonist-bound AT1(N111G) mutant receptor preferentially couples to Gq and is inadequate in beta-arrestin recruitment.

Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin.

Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alphaB-crystallin mutants in which R56 was deleted (DeltaR56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (DeltaL55 and DeltaA57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and DeltaR56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in DeltaR56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, DeltaL55 and DeltaA57, showed no significant changes in the chaperone function compared to that of the Wt. The DeltaR56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alphaB-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.

Modulation of peptidyl arginine deiminase 2 and implication for neurodegeneration.

PURPOSE: To demonstrate that elevated pressure increases the peptidyl arginine deiminase 2 (PAD2) expression in cultured astrocytes in vitro that can be modulated by pharmacological agents modulating intracellular calcium. METHODS: Isolated rat brain astrocytes were subjected to pressure treatment. Western and immunohistochemical analyses detected PAD2 protein expression. Calcium measurements were achieved employing fluorescence-based microscopic imaging and quantification system. Experiments were repeated with human optic nerve head-derived astrocytes. RESULTS: PAD2 has recently been shown to be associated with glaucomatous optic nerve. Astrocytes subjected to pressure (25-100 mmHg) show elevated level of PAD2, increased intracellular calcium, and concomitant citrullination but not significant cell death. PAD2 expression in response to elevated pressure may play a role in glaucomatous neurodegeneration. Pressure-treated astrocytes were also subjected to thapsigargin (50-250 nM) treatment, but it is unclear whether this had any further effect in increasing PAD2 expression. Conversely, treatment with calcium chelating agent BAPTA-AM (50-250 nM) results in decreased intracellular calcium concentration and PAD2. CONCLUSIONS: These results suggest calcium modulation could be exploited as therapeutic strategy to modulate pressure-induced PAD2 expression and citrullination.

Effect of site-directed mutagenesis of methylglyoxal-modifiable arginine residues on the structure and chaperone function of human alphaA-crystallin.

We reported previously that chemical modification of human alphaA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49, and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at a time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residues in mutant proteins. When compared to wild-type (wt) alphaA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6-14% and 10-14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt alphaA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49, and R103 are important determinants of the chaperone function of alphaA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation.

Glyoxalase I is critical for human retinal capillary pericyte survival under hyperglycemic conditions.

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

Dicarbonyl stress and apoptosis of vascular cells: prevention by alphaB-crystallin.

Methylglyoxal (MGO) is an alpha-dicarbonyl compound produced from triose phosphate intermediates of glycolysis. It reacts rapidly with proteins to produce advanced glycation products. We have studied the effect of MGO modification of fibronectin on retinal capillary cell viability. Our studies show that pericytes grown on MGO-modified fibronectin (FN) undergo enhanced apoptosis through the p38MAPK-mediated oxidative pathway and that alphaB-crystallin, a stress protein present in pericytes, can protect them from MGO-mediated apoptosis. Our studies with vascular endothelial cells show that hyperglycemia-induced apoptosis is inhibited by overexpression of alphaB-crystallin. These observations suggest a novel role of alphaB-crystallin in hyperglycemia-mediated damage to vascular cells in diabetes.

AlphaB-crystallin inhibits glucose-induced apoptosis in vascular endothelial cells.

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM). Transfection of pcDNA3-rat alphaB-crystallin into these cells inhibited high glucose-induced apoptosis by approximately 36%, such an effect was not observed when cells were transfected with an empty vector. AlphaB-crystallin transfection inhibited by about 35% of high glucose induced activation of caspase-3. High concentration of glucose enhanced formation of reactive oxygen species (ROS) in these cells but this was significantly (p < 0.001) curtailed by transfection of alphaB-crystallin. Results of our study indicate that alphaB-crystallin effectively inhibits both ROS formation and apoptosis in cultured vascular endothelial cells and provide a basis for future therapeutic interventions in diabetic vascular complications.

Effect of dicarbonyl modification of fibronectin on retinal capillary pericytes.

PURPOSE: To determine effects of alpha-dicarbonyl modification of an extracellular matrix protein on retinal capillary pericyte attachment and viability. METHODS: Primary cultures of bovine retinal pericytes (BRPs) were seeded on either normal fibronectin (FN) or FN modified by methylglyoxal (MGO) and glyoxal (GO). Apoptosis was measured by flow cytometry along with caspase-3 activity. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) and Akt/PKB were evaluated by Western blot analysis. Cellular glutathione and reactive oxygen species were measured. alphaB-crystallin was measured by Western blot analysis and, to determine its role in apoptosis, experiments were conducted using BRPs that were transiently transfected with alphaB-crystallin. RESULTS: Cultures seeded on MGO- or GO-modified FN showed a significant reduction in the number of viable cells, an increase in the number of apoptotic cells, and increased caspase-3 activity, which correlated with the extent of FN modification. Pericytes seeded on either type of modified FN showed phosphorylation of p38 MAPK and dephosphorylation of Akt/PKB. Cultures seeded on dicarbonyl-modified FN had reduced glutathione and increased levels of reactive oxygen species compared with those on a normal matrix. Cells on the altered matrices had reduced alphaB-crystallin levels as well. Transient transfection of rat alphaB-crystallin into BRPs significantly reduced the apoptosis triggered by alpha-dicarbonyl-modified FN. CONCLUSIONS: These observations indicate that modification of FN by alpha-dicarbonyl compounds triggers apoptosis through a combination of increased oxidative stress and reduction of alphaB-crystallin. This mechanism may contribute to loss of pericytes in diabetic retinopathy and contribute to the resultant vascular lesions.

Membrane topology and membrane retention of the ryanodine receptor calcium release channel.

The ryanodine receptor (RyR) is a Ca2+ release channel located in the sarcoplasmic/endoplasmic reticulum (ER) membrane and plays a critical role in excitation-contraction coupling of skeletal and cardiac muscles. RyR normally exists in a tetrameric structure and contains two functional domains: a carboxyl-terminal hydrophobic domain that contains the conduction pore of the Ca2+ release channel, and a large amino-terminal domain that contains sites responsible for channel regulation. Recent studies involving mutagenesis and heterologous expression have helped unravel the structure-function relationship of RyR, including transmembrane topology and intracellular localization of the Ca2+-release channel. The carboxyl-terminal portion of RyR contains the putative transmembrane segments and is sufficient to form a functional Ca2+-release channel. The amino-terminal region of the protein contains sites responsible for regulation by endogenous modulators such as Ca2+ and Mg2+ and by exogenous ligands such as caffeine. The membrane topology of RyR appears to contain an even number (four or six) of transmembrane segments with a ion selectivity filter present within a region residing between the last two segments, similar to potassium channel, whose atomic structure was described recently. The transmembrane segments also contain sequences that are responsible for localization of RyR in the endoplasmic reticulum, and this sequence is highly conserved in IP3 receptors, which also function as Ca2+-release channels.

Co-expression of MG29 and ryanodine receptor leads to apoptotic cell death: effect mediated by intracellular Ca2+ release.

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.

Activation of protein kinase C reverses capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.

Ca2+ selective ion channels of vanilloid receptor subtype-1 (TRPV1) in capsaicin-sensitive dorsal root ganglion (DRG) neurons and TRPV1 transfected Chinese hamster ovarian (CHO) cells are desensitized following calcium-dependent tachyphylaxis induced by successive applications of 100 nM capsaicin. Tachyphylaxis of TRPV1 to 100 nM capsaicin stimuli was not observed in the absence of extracellular calcium. Capsaicin sensitivity of desensitized TRPV1 ion channels recovered on application of phorbol-12-myristate-13-acetate (PMA). PMA-induced recovery of desensitized TRPV1 was primarily due to influx of extracellular calcium observed during re-application of capsaicin following desensitization. Capsazepine blocked the re-sensitization to capsaicin by PMA. Protein kinase C (PKC) inhibitory peptide PKC fragment 19-36 also inhibited re-sensitization to capsaicin by PMA. Reversal of capsaicin-induced desensitization by PMA was prevented by a mutation of TRPV1 where phosphorylation sites serine502 and serine800 were replaced with alanine. This study provides evidence for a role of PKC in reversing capsaicin-induced calcium-dependent desensitization of TRPV1 ion channels.

A retrograde signal from calsequestrin for the regulation of store-operated Ca2+ entry in skeletal muscle.

Calsequestrin (CSQ) is a high capacity Ca(2+)-binding protein present in the lumen of sarcoplasmic reticulum (SR) in striated muscle cells and has been shown to regulate the ryanodine receptor Ca(2+) release channel activity through interaction with other proteins present in the SR. Here we show that overexpression of wild-type CSQ or a CSQ mutant lacking the junction binding region (amino acids 86-191; Delta junc-CSQ) in mouse skeletal C2C12 myotube enhanced caffeine- and voltage-induced Ca(2+) release by increasing the Ca(2+) load in SR, whereas overexpression of a mutant CSQ lacking a Ca(2+) binding, aspartate-rich domain (amino acids 352-367; Delta asp-CSQ) showed the opposite effects. Depletion of SR Ca(2+) by thapsigargin initiated store-operated Ca(2+) entry (SOCE) in C2C12 myotubes. A large component of SOCE was inhibited by overexpression of wild-type CSQ or Delta junc-CSQ, whereas myotubes transfected with Delta asp-CSQ exhibited normal function of SOCE. These results indicate that the aspartate-rich segment of CSQ, under conditions of overexpression, can sustain structural interactions that interfere with the SOCE mechanism. Such retrograde activation mechanisms are possibly taking place at the junctional site of the SR.

Ca(2+)-dependent interaction between FKBP12 and calcineurin regulates activity of the Ca(2+) release channel in skeletal muscle.

Calcineurin is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse cellular functions. Here we examined the physical and functional interactions between calcineurin and ryanodine receptor (RyR) in a C2C12 cell line derived from mouse skeletal muscle. Coimmunoprecipitation experiments revealed that the association between RyR and calcineurin exhibits a strong Ca(2+) dependence. This association involves a Ca(2+) dependent interaction between calcineurin and FK506-binding protein (FKBP12), an accessory subunit of RyR. Pretreatment with cyclosporin A, an inhibitor of calcineurin, enhanced the caffeine-induced Ca(2+) release (CICR) in C2C12 cells. This effect was similar to those of FK506 and rapamycin, two drugs known to cause dissociation of FKBP12 from RyR. Overexpression of a constitutively active form of calcineurin in C2C12 cells, DeltaCnA(391-521) (deletion of the last 131 amino acids from calcineurin), resulted in a decrease in CICR. This decrease in CICR activity was partially recovered by pretreatment with cyclosporin A. Furthermore, overexpression of an endogenous calcineurin inhibitor (cain) or an inactive form of calcineurin (DeltaCnA(H101Q)) in C2C12 cells resulted in up-regulation of CICR. Taken together, our data suggest that a trimeric-interaction among calcineurin, FKBP12, and RyR is important for the regulation of the RyR channel activity and may play an important role in the Ca(2+) signaling of muscle contraction and relaxation.

Identification of a dantrolene-binding sequence on the skeletal muscle ryanodine receptor.

Dantrolene is a drug that suppresses intracellular Ca(2+) release from sarcoplasmic reticulum (SR) in skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Although its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca(2+) release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [(3)H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [(3)H]azidodantrolene, indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1 previously shown to affect RyR1 function in vitro and in vivo were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2s, peptides containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [(3)H]azidodantrolene. A monoclonal anti-RyR1 antibody that recognizes RyR1 and its 1400-amino acid N-terminal fragment recognizes DP1 and DP1-2s in both Western blots and immunoprecipitation assays and specifically inhibits [(3)H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in SR. Our results indicate that synthetic domain peptides can mimic a native, ligand-binding conformation in vitro and that the dantrolene-binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino acids 590-609.

The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.