UNIPROT:P21817 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P21817 (RyR1)
1,154 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study, we have identified calmodulin binding sequences in the skeletal muscle ryanodine receptor Ca2+ release channel. Ligand overlays on RYR fusion proteins indicate that the skeletal muscle RYR contains three calmodulin binding regions defined by residues 2937-3225, 3546-3655, and 4425-4621. The RYR fusion protein PC28 (residues 2937-3225) bound calmodulin in the presence of EGTA and Ca2+, while RYR fusion protein PC26 (residues 3546-3655) exhibited strong calmodulin binding at 10 microM Ca2+. The RYR fusion protein PC15 (residues 4425-4621) did not bind calmodulin in the presence of either EGTA or 10-50 microM Ca2+. In the presence of 100-500 microM Ca2+, the RYR fusion protein PC15 exhibited an affinity for calmodulin of approximately 50 nM. Peptides RYR1 PM2 (residues 3610-3629) and RYR1 PM3 (4534-4552) encompassing putative RYR-calmodulin binding sites were synthesized. The synthetic peptides interacted directly with dansylcalmodulin as demonstrated by their capacity to affect the fluorescence emission of dansylcalmodulin. Missense mutation analysis indicates that the Lys and Arg residues are essential for calmodulin binding to the synthetic peptide RYR1 PM3. The RYR calmodulin binding site defined by peptide PM3 lies in the myoplasmic loop 2, a few residues upstream of the putative transmembrane segment M5; the other two calmodulin binding sites are next to the putative transmembrane segments M' and M''. Thus, the effect of calmodulin on Ca2+ release might involve the regulation of the putative transmembrane segments M5, M', and M''.
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PMID:Identification and characterization of three calmodulin binding sites of the skeletal muscle ryanodine receptor. 804 9

In this study, the modification of skeletal muscle ryanodine receptor (RyR)/Ca2+-release channel with 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole (Nbd-Cl) demonstrates that lysyl residues are involved in the channel gating. Nbd-Cl was found to have a dual effect: stimulation and inhibition of ryanodine binding and single channel activities. Nbd-Cl, in a time-dependent manner, first stimulated and subsequently inhibited ryanodine binding to both membrane-bound and purified RyR. Incubation of sacroplasmic reticulum membranes with Nbd-Cl for 5-20 s resulted in enhanced ryanodine-binding activity by 2-4-fold due, to an increased binding affinity by about tenfold, with no effect on the total binding sites (Bmax). However, under prolonged incubation (5-20 min), Nbd-Cl strongly inhibited ryanodine binding by decreasing the Bmax with no effect on the binding affinity. Similar effects of stimulation and inhibition by Nbd-Cl were obtained with single channel activity of RyR reconstituted into planar lipid bilayer. Nbd-Cl initially (within a few seconds) activated the channel to a highly open state, then (within a few minutes) inactivated it to the completely closed state. Nbd-Cl-modified protein, as assayed by ryanodine binding or single channel activities, was stable against thiolysis by dithiothreitol, suggesting Nbd-Cl modification of lysyl residues. Evidence from absorption and fluorescence excitation and emission spectra also demonstrated that lysyl residues in RyR were modified by Nbd-Cl. Spectrophotometric data were used to estimate a ratio of up to 1 mol Nbd bound/mol RyR (tetramer) and up to 4 mol Nbd bound per mol RyR (tetramer) for Nbd-Cl stimulated and inhibited RyR activities, respectively. The results clearly indicate the involvement of two classes of lysyl residues in RyR activity. Modification by Nbd-Cl of the fast-reacting group led to stimulation of ryanodine binding and single channel activities, while modification of the slow-reacting group resulted in inhibition of these activities. Thus, the involvement of lysine residues in the gating of the RyR channel is proposed.
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PMID:Involvement of lysine residues in the gating of the ryanodine receptor/Ca2+-release channel of skeletal muscle sarcoplasmic reticulum. 928 20

Excitation-contraction coupling in skeletal muscle is a result of the interaction between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR). Interactions between RyR1 and DHPR are critical for the depolarization-induced activation of Ca2+ release from the sarcoplasmic reticulum, enhancement of DHPR Ca2+ channel activity, and repolarization-induced inactivation of RyR1. The DHPR III-IV loop was fused to glutathione S-transferase (GST) or His-peptide and used as a protein affinity column for 35S-labeled, in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR III-IV loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. Construction of chimeras between RyR1 and RyR2 showed that amino acids Lys954-Asp1112 retained full binding activity, whereas Leu922-Phe1075 had no binding activity. The RyR1 sequence Arg1076-Asp1112, previously shown to interact with the DHPR II-III loop (Leong, P., and MacLennan, D., H. (1998) J. Biol. Chem. 273, 7791-7794), bound to DHPR III-IV loop columns, but with only half the efficiency of binding of the longer RyR1 sequence, Lys954-Asp1112. These data suggest that the site of DHPR III-IV loop interaction contains elements from both the Lys954-Phe1075 and Arg1076-Asp1112 fragments. The presence of 4 +/- 0.4 microM GST-DHPR II-III or 5 +/- 0.1 microM His-peptide-DHPR III-IV was required for half-maximal co-purification of 35S-labeled RyR1 Leu922-Asp1112 on glutathione-Sepharose or Ni2+-nitrilotriacetic acid. Dose-dependent inhibition of 35S-labeled RyR1 Leu922-Asp1112 binding to GST-DHPR II-III and GST-DHPR III-IV by His10-DHPR II-III and His-peptide-DHPR III-IV was observed. These studies indicate that the DHPR II-III and III-IV loops bind to contiguous and possibly overlapping sites on RyR1 between Lys 954 and Asp1112.
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PMID:The cytoplasmic loops between domains II and III and domains III and IV in the skeletal muscle dihydropyridine receptor bind to a contiguous site in the skeletal muscle ryanodine receptor. 979 15

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.
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PMID:Excitation-contraction coupling is not affected by scrambled sequence in residues 681-690 of the dihydropyridine receptor II-III loop. 1091 79

Both imperatoxin A (IpTx(a)), a 33-residue peptide toxin from scorpion venom, and peptide A, derived from the II-III loop of dihydropyridine receptor (DHPR), interact specifically with the skeletal ryanodine receptor (RyR1), which is a Ca(2+)-release channel in the sarcoplasmic reticulum, but with considerably different affinities. IpTx(a) activates RyR1 with nanomolar affinity, whereas peptide A activates RyR1 at micromolar concentrations. To investigate the molecular basis for high-affinity activation of RyR1 by IpTx(a), we have determined the NMR solution structure of IpTx(a), and identified its functional surface by using alanine-scanning analogues. A detailed comparison of the functional surface profiles for two peptide activators revealed that IpTx(a) exhibits a large functional surface area (approx. 1900 A(2), where 1 A=0.1 nm), based on a short double-stranded antiparallel beta-sheet structure, while peptide A bears a much smaller functional surface area (approx. 800 A(2)), with the five consecutive basic residues (Arg(681), Lys(682), Arg(683), Arg(684) and Lys(685)) being clustered at the C-terminal end of the alpha-helix. The functional surface of IpTx(a) is composed of six essential residues (Leu(7), Lys(22), Arg(23), Arg(24), Arg(31) and Arg(33)) and several other important residues (His(6), Lys(8), Arg(9), Lys(11), Lys(19), Lys(20), Gly(25), Thr(26), Asn(27) and Lys(30)), indicating that amino acid residues involved in RyR1 activation make up over the half of the toxin molecule with the exception of cysteine residues. Taken together, these results suggest that the site where peptide A binds to RyR1 belongs to a subset of macrosites capable of being occupied by IpTx(a), resulting in differing the affinity and the mode of activation.
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PMID:Molecular basis of the high-affinity activation of type 1 ryanodine receptors by imperatoxin A. 1453 45

Buthotus judaicus toxin 1 (BjTx-1) and toxin 2 (BjTx-2), two novel peptide activators of ryanodine receptors (RyR), were purified from the venom of the scorpion B. judaicus. Their amino acid sequences differ only in 1 residue out of 28 (residue 16 corresponds to Lys in BjTx-1 and Ile in BjTx-2). Despite a slight difference in EC(50), both toxins increased binding of [(3)H]ryanodine to skeletal sarcoplasmic reticulum at micromolar concentrations but had no effect on cardiac or liver microsomes. Their activating effect was Ca(2+)-dependent and was synergized by caffeine. B. judaicus toxins also increased binding of [(3)H]ryanodine to the purified RyR1, suggesting that a direct protein-protein interaction mediates the effect of the peptides. BjTx-1 and BjTx-2 induced Ca(2+) release from Ca(2+)-loaded sarcoplasmic reticulum vesicles in a dose-dependent manner and induced the appearance of long lived subconductance states in skeletal RyRs reconstituted into lipid bilayers. Three-dimensional structural modeling reveals that a cluster of positively charged residues (Lys(11) to Lys(16)) is a prominent structural motif of both toxins. A similar structural motif is believed to be important for activation of RyRs by imperatoxin A (IpTx(a)), another RyR-activating peptide (Gurrola, G. B., Arevalo, C., Sreekumar, R., Lokuta, A. J., Walker, J. W., and Valdivia, H. H. (1999) J. Biol. Chem. 274, 7879-7886). Thus, it is likely that B. judaicus toxins and imperatoxin A bind to RyRs by means of electrostatic interactions that lead to massive conformational changes in the channel protein. The different affinity and structural diversity of this family of scorpion peptides makes them excellent peptide probes to identify RyR domains that trigger the channel to open.
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PMID:Activation of skeletal ryanodine receptors by two novel scorpion toxins from Buthotus judaicus. 1506 3

In the present work, we purified and characterized a novel toxin named hemicalcin from the venom of the Iranian chactoid scorpion Hemiscorpius lepturus where it represents 0.6% of the total protein content. It is a 33-mer basic peptide reticulated by three disulfide bridges, and that shares between 85 and 91% sequence identity with four other toxins, all known or supposed to be active on ryanodine-sensitive calcium channels. Hemicalcin differs from these other toxins by seven amino acids at positions 9 (leucine/arginine), 12 (alanine/glutamic acid), 13 (aspartic acid/asparagine), 14 (lysine/asparagine), 18 (serine/glycine), 26 (threonine/alanine) and 28 (proline/isoleucine/alanine). In spite of these differences, hemicalcin remains active on ryanodine-sensitive Ca2+ channels, since it increases [3H]ryanodine binding on RyR1 (ryanodine receptor type 1) and triggers Ca2+ release from sarcoplasmic vesicles. Bilayer lipid membrane experiments, in which the RyR1 channel is reconstituted and its gating properties are analysed, indicate that hemicalcin promotes an increase in the opening probability at intermediate concentration and induces a long-lasting subconductance level of 38% of the original amplitude at higher concentrations. Mice intracerebroventricular inoculation of 300 ng of hemicalcin induces neurotoxic symptoms in vivo, followed by death. Overall, these data identify a new biologically active toxin that belongs to a family of peptides active on the ryanodine-sensitive channel.
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PMID:Hemicalcin, a new toxin from the Iranian scorpion Hemiscorpius lepturus which is active on ryanodine-sensitive Ca2+ channels. 1729 Nov 97

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.
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PMID:Interaction of the Lys(3614)-Asn(3643) calmodulin-binding domain with the Cys(4114)-Asn(4142) region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation. 1817 25

The 33 amino acid scorpion toxin maurocalcine (MCa) has been shown to modify the gating of the skeletal-type ryanodine receptor (RyR1). Here we explored the effects of MCa and its mutants ([Ala(8)]MCa, [Ala(19)]MCa, [Ala(20)]MCa, [Ala(22)]MCa, [Ala(23)]MCa, and [Ala(24)]MCa) on RyR1 incorporated into artificial lipid bilayers and on elementary calcium release events (ECRE) in rat and frog skeletal muscle fibers. The peptides induced long-lasting subconductance states (LLSS) on RyR1 that lasted for several seconds. However, their average length and frequency were decreased if the mutation was placed farther away in the 3D structure from the critical (24)Arg residue. The effect was strongly dependent on the direction of the current through the channel. If the direction was similar to that followed by calcium during release, the peptides were 8- to 10-fold less effective. In fibers long-lasting calcium release events were observed after the addition of the peptides. The average length of these events correlated well with the duration of LLSS. These data suggest that the effect of the peptide is governed by the large charged surface formed by residues Lys(20), Lys(22), Arg(23), Arg(24), and Lys(8). Our observations also indicate that the results from bilayer experiments mimic the in situ effects of MCa on RyR1.
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PMID:Charged surface area of maurocalcine determines its interaction with the skeletal ryanodine receptor. 1862 23

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca(2+) release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca(2+)-S100A1 competes with Ca(2+)-calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca(2+)-S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616-3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca(2+)-S100A1. These same residues were shown previously to be important for RyR1 binding to Ca(2+)-CaM. A model for regulating muscle contraction is presented in which Ca(2+)-S100A1 and Ca(2+)-CaM compete directly for the same binding site on the ryanodine receptor.
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PMID:S100A1 and calmodulin compete for the same binding site on ryanodine receptor. 1865 Apr 34

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