Ca2+ binding sites of the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum. Low affinity binding site(s) as probed by terbium fluorescence.

Hadad, Nurit; Zable, Anthony C.; Abramson, Jonathan J.; Shoshan‐Barmatz, Varda

doi:10.1016/s0021-9258(17)31470-9

Cited by 23 publications

(6 citation statements)

References 35 publications

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“…[ 3 H]Ryanodine Binding Ryanodine binding by SR membranes was assayed as described previously [10]. Membranes were incubated for 60 min at 37ºC in a standard binding solution containing 1 M NaCl, 20 mM Mops (pH 7.4), 50 mM free [Ca 2+ ], and 20 nM [ 3 H]ryanodine, followed by vacuum filtration of the sample through nitrocellulose filters (0.3 mm).…”

Section: Mitochondrial Swellingmentioning

confidence: 99%

“…Extensive studies have focused on developing specific Ca 2+ binding reagents for probing changes in [Ca 2+ ]i during cell function; fewer efforts, however, have been made to probe Ca 2+ binding sites in proteins. In addition, despite advances in defining Ca 2+ binding proteins, considerable experimental difficulties still remain in locating their Ca 2+ binding sites [10].…”

Section: Introductionmentioning

confidence: 99%

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A Photoactivable Probe for Calcium Binding Proteins

Israelson

Arzoine

Abu-Hamad

et al. 2005

Chemistry & Biology

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Ca2+ as a signaling molecule carries information pivotal to cell life and death via its reversible interaction with a specific site in a protein. Although numerous Ca2+-dependent activities are known, the proteins responsible for some of these activities remain unidentified. We synthesized and characterized a photoreactive reagent, azido ruthenium (AzRu), which interacts specifically with Ca2+ binding proteins and strongly inhibits their Ca2+-dependent activities, regardless of their catalytic mechanisms or functional state as purified proteins, embedded in the membrane or in intact cells. As expected from a Ca2+ binding protein-specific reagent, AzRu had no effect on Ca2+-independent and Mg2+-dependent activities. Az103Ru covalently bound, and specifically labeled, known Ca2+ binding proteins. AzRu is a photoreactive reagent that provides an approach for identification of Ca2+ binding proteins, characterization of their binding sites, and exploration of new Ca2+-dependent processes.

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Section: Mitochondrial Swellingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Photoactivable Probe for Calcium Binding Proteins

Israelson

Arzoine

Abu-Hamad

et al. 2005

Chemistry & Biology

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“…Due to their similarity in ionic radii, coordination geometry and donor atom preference (Martin and Richardson 1979), lanthanide ions can be used to determine calcium binding sites in proteins. Lanthanides have been shown to interact with various Ca 2+binding proteins (Hadad et al 1994;Reed and Bygrave 1974) and VDAC1, for which they induce channel closure and inhibit Ca 2+ transport (Gincel et al 2001). An advantage of paramagnetic lanthanide ions like Gd 3+ is that they induce paramagnetic relaxation enhancement (PRE), such that direct binding can be established from resonance broadening and can be distinguished from structural changes in the protein.…”

Section: Lanthanide Ions Confirm the C-terminal Ca 2+ -Interacting Re...mentioning

confidence: 99%

“…Another alternative for probing of calcium binding sites is the dye ruthenium red (RuR), which binds to Ca 2+ -binding sites of other proteins (Hadad et al 1994;Reed and Bygrave 1974), induces channel closure of wt VDAC1 reconstituted in bilayers, and protects cells from apoptosis (Israelson et al 2008). Furthermore, CaCl 2 competes with the binding of azo ruthenium red, a photoreactive ruthenium compound, and E73 and E203 have been implicated in binding of RuR as well (Israelson et al 2007).…”

Section: Interaction With Ruthenium Red (Rur) Confirms Ca 2+ Interact...mentioning

confidence: 99%

“…Two glutamic acids, E73 and E203, were suggested to be important for Ca 2+binding, since mutation of either glutamic acid abolished binding of RuR and azo ruthenium red (AzRu) to VDAC1 (Israelson et al 2007;Israelson et al 2008). RuR is known to bind to Ca 2+ -binding sites of other proteins (Hadad et al 1994;Reed and Bygrave 1974) and CaCl 2 competes with AzRu binding (Israelson et al 2007). RuR induced chemical shift and intensity changes in similar regions in the N-and C-terminus (Figure 42), supporting the presence of both Ca 2+ binding sites.…”

Section: Vdac-atp-hexokinase: a Modelmentioning

confidence: 99%

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Dynamics and interactions of the voltage-dependent anion channel 1 studied by NMR spectroscopy

Villinger¹

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The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, acts as a gatekeeper for the entry and exit of mitochondrial metabolites and is involved in mitochondrial apoptosis. Recent determination of its high-resolution structure by three independent groups revealed a 19-stranded β-barrel with a differently arranged N-terminal α-helix inside the pore.In this thesis, the NMR resonance assignment of isoform one of human VDAC (VDAC1) in solution is increased. In addition, this study provides evidence for a kinked α-helical structure of the N-terminus, which is compatible with the crystal structure of VDAC1, although other structures cannot be excluded. Furthermore, this study reveals functional dynamics of VDAC1 by a combination of solution NMR spectroscopy, Gaussian network model (GNM) analysis, and molecular dynamics (MD) simulations.Low signal intensities indicate conformational exchange in the second part of the Nterminal α-helix and the linker connecting the α-helix to the first β-strand. In addition, mutation of arginine 15 in the second α-helical part affects the stability of the α-helix and the overall barrel in a complex manner. Micro-to millisecond dynamics are significantly increased in the N-terminal α-helix, the linker, and the N-terminal six βstrands of VDAC1 in micellar solution. In addition, hydrogen bonds are instable in the N-terminal three β-strands. In agreement, the N-terminal β-strands exhibit increased Bfactors in the crystal structure of VDAC1 and intrinsic instability predicted by the GNM analysis. Mutation or chemical modification of the membrane facing glutamic acid 73 (E73) strongly reduces the micro-to millisecond dynamics in solution. MD simulations reveal that a charge on E73 accounts for the elevation of N-terminal protein dynamics as well as a thinning of the nearby membrane. Since E73 is necessary for hexokinase-Iinduced VDAC1 channel closure and inhibition of apoptosis, these results imply that micro-to millisecond dynamics in the N-terminal part of the β-barrel are essential for VDAC1 interaction and gating. Moreover, the data suggest that dynamics in the α-helix are connected with these processes. III Furthermore, this study reveals two binding sites for the pore's most important transport substrate ATP. The location of the ATP binding sites, one of them comprising the N-terminal α-helix, the linker, and nearby β-strands, suggests controlled metabolite flux and ligand-induced stabilization of the open state of the VDAC1 pore. Finally, Ca 2+ is found to interact with two distinct N-terminal and C-terminal regions in the β-barrel. These regions overlap with VDAC1 oligomerization sites and dynamic regions, suggesting a connection between Ca 2+ interaction, gating, and oligomerization. Zusammenfassung Der spannungsabhängige Anionenkanal (engl. "voltage-dependent anion channel", VDAC), das häufigste Protein in der äußeren Mitochondrienmembran, dient als wichtiger Kontrollpunkt für den Ein-und Austritt von mitochondrialen Metaboliten und ...

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Binding Sites for Ca²⁺‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

et al. 1996

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The calcium channel and the 'calcium release channel' of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0.1 and 300 pg m1-l. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) C3H]isradipine (PN 200-1 lo), the phenylalkylamine [3H]verapamil and the alkaloid C3H]ryanodine. Preliminary binding studies with the benzothiazepine C3H]diltiazem suggest a low-affinity binding site with a IC5, value of 3.3 PM. All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 p~. EGTA at 10 p~ abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca2+ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 p~ EGTA. With respect to high Ca2+ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73,40 and 20%, respectively, at 5 mM Ca2+.Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K , values of 0.95 nM (B,,, = 550 fmol mg-' protein) and 0.75 nM (B,,, = 213 fmol mg-' protein) were determined respectively. A loweraffinity binding site was identified in binding studies with verapamil (K, = 7.4 nM and B , = 27 fmol mg-' protein). ['Hlisradipine displacement studies with several dihydropyridines revealed the following ranking of affinity : nitrendipine > isradipine > Bay K8664 9 nicardipine. Displacement of [3H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by ( f )verapamil, nor-methylverapamil and R( + )verapamil.

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Ca2+ binding sites of the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum. Low affinity binding site(s) as probed by terbium fluorescence.

Cited by 23 publications

References 35 publications

A Photoactivable Probe for Calcium Binding Proteins

A Photoactivable Probe for Calcium Binding Proteins

Dynamics and interactions of the voltage-dependent anion channel 1 studied by NMR spectroscopy

Binding Sites for Ca²⁺‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

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Ca2+ binding sites of the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum. Low affinity binding site(s) as probed by terbium fluorescence.

Cited by 23 publications

References 35 publications

A Photoactivable Probe for Calcium Binding Proteins

A Photoactivable Probe for Calcium Binding Proteins

Dynamics and interactions of the voltage-dependent anion channel 1 studied by NMR spectroscopy

Binding Sites for Ca2+‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides

Contact Info

Product

Resources

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Binding Sites for Ca²⁺‐Channel Effectors and Ryanodine inPeriplaneta americana—Possible Targets for New Insecticides