Jianjie Ma

Mitochondrial Depolarization Accompanies Cytochrome c Release During Apoptosis in PC6 Cells

Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester decreased abruptly in individual cells from 2 to 7 h after treatment with staurosporine. Depolarization was accompanied by cytochromec release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The results show that mitochondrial depolarization accompanies cytochromec release in pheochromocytoma-6 cells undergoing apoptosis.

Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor

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.

Identification of a Dantrolene-binding Sequence on the Skeletal Muscle Ryanodine Receptor

Dantrolene is a drug that suppresses intracellular Ca2+ 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 Ca2+ release channel in SR, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene. Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]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 andin 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 [3H]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 [3H]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 asp‐rich region at the carboxyl‐terminus of calsequestrin binds to Ca2+ and interacts with triadin

Calsequestrin (CSQ) is a high capacity Ca2+ binding protein in the junctional sarcoplasmic reticulum of striated muscles, and has been shown to regulate the ryanodine receptor (RyR) through triadin and junctin. In order to identify the functional roles of specific regions on CSQ, several CSQ deletion mutants were prepared by molecular cloning and Escherichia coli expression. 45Ca2+ overlay assay using a native gel system revealed that the major Ca2+ binding motif of CSQ resides in the asp‐rich region (amino acids 354–367). In an in vitro binding assay using a glutathione‐S‐transferase affinity column, the interaction between CSQ and triadin was found to be Ca2+‐dependent, and the site of interaction was confined to the asp‐rich region of CSQ. Our results suggest that the asp‐rich region of CSQ could participate in the RyR‐mediated Ca2+ release process by offering a direct binding site to luminal Ca2+ as well as triadin.

Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel.

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, ΔR(708–835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5′-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the ΔR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the ΔR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the ΔR-CFTR channel results;P o increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.

Molecular Cloning of cDNA Encoding a Drosophila Ryanodine Receptor and Functional Studies of the Carboxyl-Terminal Calcium Release Channel

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.

A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions.

Direct Activation of Cystic Fibrosis Transmembrane Conductance Regulator Channels by 8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-Diallyl-8-cyclohexylxanthine (DAX)

8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (ΔF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy.

Junctophilin-2 Expression Silencing Causes Cardiocyte Hypertrophy and Abnormal Intracellular Calcium-Handling

Background—Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca2+) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca2+ channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca2+ handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca2+ handling in a prohypertrophic manner. Methods and Results—JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca2+-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca2+ transient amplitudes that were insensitive to L-type Ca2+ channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca2+ levels were observed with potentially increased total Ca2+ stores. Spontaneous Ca2+ oscillations were elicited at a higher extracellular [Ca2+] and with decreased frequency in JPH2 knockdown cells. Conclusions—Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca2+ homeostasis, which may be associated with prohypertrophic remodeling.

Compromised store-operated Ca2+ entry in aged skeletal muscle

In aged skeletal muscle, changes to the composition and function of the contractile machinery cannot fully explain the observed decrease in the specific force produced by the contractile machinery that characterizes muscle weakness during aging. Since modification in extracellular Ca2+ entry in aged nonexcitable and excitable cells has been recently identified, we evaluated the functional status of store‐operated Ca2+ entry (SOCE) in aged mouse skeletal muscle. Using Mn2+ quenching of Fura‐2 fluorescence and confocal‐microscopic imaging of Ca2+ movement from the transverse tubules, we determined that SOCE was severely compromised in muscle fibers isolated from aged mice (26–27 months) as compared with those from young (2–5 months) mice. While reduced SOCE in aged skeletal muscle does not appear to result from altered expression levels of STIM1 or reduced expression of mRNA for Orai, this reduction in SOCE is mirrored in fibers isolated from young mice null for mitsugumin‐29, a synaptophysin‐related protein that displays decreased expression in aged skeletal muscle. Our data suggest that decreased mitsugumin‐29 expression and reduced SOCE may contribute to the diminished intracellular Ca2+ homeostatic capacity generally associated with muscle aging.