Identification of a key determinant of ryanodine receptor type 1 required for activation by 4-chloro-m-cresol

4-Chloro-m-cresol (4-CmC) is a potent and specific activator of the intracellular Ca2+ release channel, the ryanodine receptor (RyR). We have previously shown that RyR1 expressed in dyspedic 1B5 myotubes is activated by 4-CmC, whereas RyR3 is not (Fessenden, J. D., Wang, Y., Moore, R. A., Chen, S. R. W., Allen, P. D., and Pessah, I. N. (2000) Biophys. J. 79, 2509-2525). To identify region(s) on RyR1 that are responsible for mediating activation by 4-CmC, we expressed RyR1-RyR3 chimeric proteins in dyspedic 1B5 myotubes and then measured 4-CmC-induced increases in intracellular Ca2+. Substitution of the C-terminal third of RyR1 into RyR3 imparted 4-CmC sensitivity to the resulting chimera, thus suggesting that determinants required for activation by 4-CmC are located in this region. We subdivided the C-terminal third of RyR1 into smaller segments and identified two overlapping regions of RyR1 (amino acids 3769-4180 and 4007-4382) that each imparted 4-CmC sensitivity to RyR3. Substitution of the 173 amino acids of RyR1 common to these two chimeras (amino acids 4007-4180) also weakly restored 4-CmC sensitivity in the resulting chimera. To confirm these findings, we created a complementary set of chimeras containing RyR3 substitutions in RyR1. Substitution of the RyR3 C terminus into RyR1 disrupted 4-CmC sensitivity in the resulting chimera. In addition, substitution of the corresponding RyR3 sequence into positions 4007-4180 of RyR1 disrupted 4-CmC sensitivity. Taken together, these results suggest that essential determinants required for activation of RyR1 by 4-CmC reside within a 173-amino acid region between residues 4007 and 4180.     

N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

Factor XIIIa mediated attachment of S. aureus fibronectin-binding protein A (FnbA) to fibrin: identification of Gln103 as a major cross-linking site

In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.     

Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca(2+) release channel (ryanodine receptor) activity and conductance

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid residue I4897 homologous to the selectivity filter of K(+) channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not absolutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K(+) and Ca(2+) conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.     

Calmodulin oxidation and methionine to glutamine substitutions reveal methionine residues critical for functional interaction with ryanodine receptor-1

Calmodulin (CaM) binds to the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) with high affinity, and it may act as a Ca(2+)-sensing subunit of the channel. Apo-CaM increases RyR1 channel activity, but Ca(2+)-CaM is inhibitory. Here we examine the functional effects of CaM oxidation on RyR1 regulation by both apo-CaM and Ca(2+)-CaM, as assessed via determinations of [(3)H]ryanodine and [(35)S]CaM binding to skeletal muscle sarcoplasmic reticulum vesicles. Oxidation of all nine CaM Met residues abolished functional interactions of CaM with RyR1. Incomplete CaM oxidation, affecting 5-8 Met residues, increased the CaM concentration required to modulate RyR1, having a greater effect on the apo-CaM species. Mutating individual CaM Met residues to Gln demonstrated that Met-109 was required for apo-CaM activation of RyR1 but not for Ca(2+)-CaM inhibition of the channel. Furthermore, substitution of Gln for Met-124 increased the apo- and Ca(2+)-CaM concentrations required to regulate RyR1. These results thus identify Met residues critical for the productive association of CaM with RyR1 channels and suggest that oxidation of CaM may contribute to altered regulation of sarcoplasmic reticulum Ca(2+) release during oxidative stress.     

Different regions in skeletal and cardiac muscle ryanodine receptors are involved in transducing the functional effects of calmodulin

Calmodulin (CaM) inhibits the skeletal muscle ryanodine receptor-1 (RyR1) and cardiac muscle RyR2 at micromolar Ca(2+) but activates RyR1 and inhibits RyR2 at submicromolar Ca(2+) by binding to a single, highly conserved CaM-binding site. To identify regions responsible for the differential regulation of RyR1 and RyR2 by CaM, we generated chimeras encompassing and flanking the CaM-binding domain. We found that the exchange of the N- and C-terminal flanking regions differentially affected RyR1 and RyR2. A RyR1/RyR2 chimera with an N-terminal flanking RyR2 substitution (RyR2 amino acid (aa) 3537-3579) was activated by CaM in single channel measurements at both submicromolar and micromolar Ca(2+). A RyR2/RyR1 chimera with a C-terminal flanking the 86-amino acid RyR1 substitution (RyR1 aa 3640-3725) bound (35)S-CaM but was not inhibited by CaM at submicromolar Ca(2+). In this region, five non-conserved amino acid residues (RyR1 aa 3680 and 3682-3685 and RyR2 aa 3647 and 3649-3652) differentially affect RyR helical probability. Substitution of the five amino acid residues in RyR1 with those of RyR2 showed responses to CaM comparable with wild type RyR1. In contrast, substitution of the five amino acid residues in RyR2 with those of RyR1 showed loss of CaM inhibition, whereas substitution of the five RyR2 sequence residues in the RyR2 chimera containing the RyR1 calmodulin-binding domain and C-flanking sequence restored wild type RyR2 inhibition by CaM at submicromolar Ca(2+). The results suggest that different regions are involved in CaM modulation of RyR1 and RyR2. They further suggest that five non-conserved amino acids in the C-terminal region flanking the CaM-binding domain have a key role in CaM inhibition of RyR2.     

The calmodulin binding region of the skeletal ryanodine receptor acts as a self-modulatory domain

A synthetic peptide (CaMBP) matching amino acids 3614-3643 of the skeletal ryanodine receptor (RyR1) binds to both Ca2+-free calmodulin (CaM) and Ca2+-bound CaM with nanomolar affinity [J. Biol. Chem. 276 (2001) 2069]. We report here that CaMBP increases [3H]ryanodine binding to RyR1 in a dose- and Ca2+-dependent manner; it also induces Ca2+ release from SR vesicles, and increases open probability (P(o)) of single RyR channels reconstituted in planar lipid bilayers. Further, CaMBP removes CaM associated with SR vesicles and increases [3H]ryanodine binding to purified RyR1, suggesting that its mechanism of action is two-fold: it removes endogenous inhibitors and also interacts directly with complementary regions in RyR1. Remarkably, the N-terminus of CaMBP activates RyRs while the C-terminus of CaMBP inhibits RyR activity, suggesting the presence of two discrete functional subdomains within this region. A ryr1 mutant lacking this region, RyR1-Delta3614-3643, was constructed and expressed in dyspedic myoblasts (RyR1-knockout). The depolarization-, caffeine- and 4-chloro-m-cresol (4-CmC)-induced Ca2+ transients in these cells were dramatically reduced compared with cells expressing wild type RyR1. Deletion of the 3614-3643 region also resulted in profound changes in unitary conductance and channel gating. We thus propose that the RyR1 3614-3643 region acts not only as the CaM binding site, but also as an important modulatory domain for RyR1 function.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.     

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.     

Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

Ryanodine receptors (RyRs) are large tetrameric calcium (Ca^2 +) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca^2 + sensitivity, propagating sarcoplasmic reticulum luminal Ca^2 + release in the process of excitation–contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1–559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R169Q and delta exon 3 (Δ3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile α-helix, unique to type 2; further, this α2 helix rescues the β-strand lost in RyR2A Δ3 but remains dynamic in the hot-spot loop (HS-loop) P164S, R169Q and R176Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A α2 helix is in close proximity to dense “columns” projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain.     

Endoplasmic reticulum Ca2+ measurements reveal that the cardiac ryanodine receptor mutations linked to cardiac arrhythmia and sudden death alter the threshold for store-overload-induced Ca2+ release

A number of RyR2 (cardiac ryanodine receptor) mutations linked to ventricular arrhythmia and sudden death are located within the last C-terminal approximately 500 amino acid residues, which is believed to constitute the ion-conducting pore and gating domain of the channel. We have previously shown that mutations located near the C-terminal end of the predicted TM (transmembrane) segment 10, the inner pore helix, can either increase or decrease the propensity for SOICR (store-overload-induced Ca2+ release), also known as spontaneous Ca2+ release. In the present study, we have characterized an RyR2 mutation, V4653F, located in the loop between the predicted TM 6 and TM 7a, using an ER (endoplasmic reticulum)-targeted Ca2+-indicator protein (D1ER). We directly demonstrated that SOICR occurs at a reduced luminal Ca2+ threshold in HEK-293 cells (human embryonic kidney cells) expressing the V4653F mutant as compared with cells expressing the RyR2 wild-type. Single-channel analyses revealed that the V4653F mutation increased the sensitivity of RyR2 to activation by luminal Ca2+. In contrast with previous reports, the V4653 mutation did not alter FKBP12.6 (FK506-binding protein 12.6 kDa; F506 is an immunosuppressant macrolide)-RyR2 interaction. Luminal Ca2+ measurements also showed that the mutations R176Q/T2504M, S2246L and Q4201R, located in different regions of the channel, reduced the threshold for SOICR, whereas the A4860G mutation, located within the inner pore helix, increased the SOICR threshold. We conclude that the cytosolic loop between TM 6 and TM 7a plays an important role in determining the SOICR threshold and that the alteration of the threshold for SOICR is a common mechanism for RyR2-associated ventricular arrhythmia.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

Intersegment hydrogen bonds as possible structural determinants of the N/Q/R site in glutamate receptors.

Specific electrophysiological and pharmacological properties of ionic channels in NMDA, AMPA, and kainate subtypes of ionotropic glutamate receptors (GluRs) are determined by the Asn (N), Gln (Q), and Arg (R) residues located at homologous positions of the pore-lining M2 segments (the N/Q/R site). Presumably, the N/Q/R site is located at the apex of the reentrant membrane loop and forms the narrowest constriction of the pore. Although the shorter Asn residues are expected to protrude in the pore to a lesser extent than the longer Gln residues, the effective dimension of the NMDA channel (corresponding to the size of the largest permeant organic cation) is, surprisingly, smaller than that of the AMPA channel. To explain this paradox, we propose that the N/Q/R residues form macrocyclic structures (rings) stabilized by H-bonds between a NH(2) group in the side chain of a given M2 segment and a C==O group of the main chain in the adjacent M2 segment. Using Monte Carlo minimization, we have explored conformational properties of the rings. In the Asn, but not in the Gln ring, the side-chain oxygens protruding into the pore may facilitate ion permeation and accept H-bonds from the blocking drugs. In this way, the model explains different electrophysiological and pharmacological properties of NMDA and non-NMDA GluR channels. The ring of H-bonded polar residues at the pore narrowing resembles the ring of four Thr(75) residues observed in the crystallographic structure of the KcsA K(+) channel.     

Non-specific effects of 4-chloro-m-cresol may cause calcium flux and respiratory burst in human neutrophils

We examined the effects of 4-chloro-m-cresol (4-CmC, a potent and specific activator of ryanodine receptors) on Ca(2+)-release/influx and respiratory burst in freshly isolated human PMN as well as HL60 cells. 4-CmC induces Ca(2+) store-depletion in a dose-dependent manner at concentrations between 400muM and 3mM, however no dose-dependent effect on Ca(2+)-influx was found. 4-CmC depleted Ca(2+) stores that were shared with the GPC agonists such as fMLP and PAF, and therefore 4-CmC presumably depletes Ca(2+) from ER. Since the authentic ligand for RyR is cyclic ADP-ribose (cADPR), we assessed the functional relevance of RyR in PMN by studying the presence and function of membrane-bound ADP-ribosyl cyclase (CD38) in PMN. First, expression of CD38 was confirmed by RT-PCR using cDNA from HL60 cells. Second, PMN from trauma patients showed significantly enhanced CD38 expression than those from healthy volunteers. In addition, although no chemotaxis effect was detected by 4-CmC, it stimulated respiratory burst in PMN in a dose-dependent manner. Our findings suggest that RyRs exist in human PMN and that RyR pathway may play an active role in inflammatory PMN calcium signaling. 8-Br-cADPR and cyclic 3-deaza-ADP did not have inhibitory effects either on 4-CmC-induced Ca(2+) store-depletion or on respiratory burst, on the other hand, PLC inhibitor, U73122, completely attenuated both 4-CmC-induced Ca(2+) store-depletion and respiratory burst. Although it has been used as a specific activator of RyR, 4-CmC has non-specific effects which cause Ca(2+) store-depletion and respiratory burst at least in human PMN.     

Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.     

Role of calmodulin methionine residues in mediating productive association with cardiac ryanodine receptors

Calmodulin (CaM) binds to the cardiac ryanodine receptor Ca2+ release channel (RyR2) with high affinity and may act as a regulatory channel subunit. Here we determine the role of CaM Met residues in the productive association of CaM with RyR2, as assessed via determinations of [3H]ryanodine and [35S]CaM binding to cardiac muscle sarcoplasmic reticulum (SR) vesicles. Oxidation of all nine CaM Met residues abolished the productive association of CaM with RyR2. Substitution of the COOH-terminal Mets of CaM with Leu decreased the extent of CaM inhibition of cardiac SR (CSR) vesicle [3H]ryanodine binding. In contrast, replacing the NH2-terminal Met of CaM with Leu increased the concentration of CaM required to inhibit CSR [3H]ryanodine binding but did not alter the extent of inhibition. Site-specific substitution of individual CaM Met residues with Gln demonstrated that Met124 was required for both high-affinity CaM binding to RyR2 and for maximal CaM inhibition. These results thus identify a Met residue critical for the productive association of CaM with RyR2 channels.     

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.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.