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The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.

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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

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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-Daspartic 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 cannabi-,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 antagonistsIt is interesting to note that a transient Ca 2ϩ signal was observed after the acute application of WIN. This Ca 2ϩ increase was blocked by a CB1 receptor antagonist AM251, IP 3 receptor antagonist 2-aminoethyl diphenylborinate, or by depleting intracellular Ca 2ϩ stores with the endoplasmic reticulum Ca 2ϩ pump inhibitor thapsigargin. Removal of extracellular Ca 2ϩ , on the other hand, had no effect on the CB1 receptor-induced Ca 2ϩ 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 2ϩ from intracellular stores, raises the cytosolic Ca 2ϩ levels, and inhibits the NMDAmediated Ca 2ϩ influx and cell death through a process that remains to be determined.Cannabinoid receptors are members of the superfamily of G i /G o -coupled receptors and include at least two subtypes, Cannabinoid receptor-1 (CB1) and CB2 receptors. The CB1 receptor is expressed primarily in the central nervous system (Matsuda et al., 1990) and peripheral nociceptors (Agarwal et al., 2007), whereas the CB2 receptor is predominantly expressed in immune cells (Munro et al., 1993) and is also detectable in brainstem neurons (Van Sickle et al., 2005) and spinal cord . More recently, another cannabinoid receptor GPR55 was identified that appears to be highly expressed in large dorsal root ganglion neurons

The mechanism by which transforming growth factor-␤1 (TGF-␤1) induces apoptosis of prostate epithelial cells was studied in the NRP-154 rat prostate epithelial cell line. TGF-␤1 down-regulates expression of Bcl-xL and poly(ADP-ribosyl)polymerase (PARP), promotes cytochrome c release, up-regulates expression of latent caspase-3, and activates caspases 3 and 9. We tested the role of Bcl-xL in this cascade by stably overexpressing Bcl-xL to prevent loss by TGF-␤1. Clones overexpressing Bcl-xL are resistant to TGF-␤1 with respect to induction of apoptosis, cytochrome c release, activation of caspases 9 and 3, and cleavage of PARP; yet they remain sensitive to TGF-␤1 by cell cycle arrest, induction of both fibronectin and latent caspase-3 expression, and loss of PARP expression. We show that Bcl-xL associates with Apaf-1 in NRP-154 cells; but this association does not inhibit the activation of caspases 9 and 3 by cytochrome c. Together, our data suggest that TGF-␤1 induces apoptosis through loss of Bcl-xL, leading to cytochrome c release and the subsequent activation of caspases 9 and 3. Moreover, our data demonstrate that the antiapoptotic effect of Bcl-xL occurs by inhibition of mitochondrial cytochrome c release and not through antagonizing Apaf-1-dependent processing of caspases 9 and 3.

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.