Structural and functional interactions between the EF hand domain and S2-S3 loop in the type-1 ryanodine receptor ion channel.

Previous cryo-electron micrographs suggested that the skeletal muscle Ca2+ release channel, ryanodine receptor (RyR)1, is regulated by intricate interactions between the EF hand Ca2+ binding domain and the cytosolic loop (S2-S3 loop). However, the precise molecular details of these interactions and functional consequences of the interactions remain elusive. Here, we used molecular dynamics simulations to explore the specific amino acid pairs involved in hydrogen bond interactions within the EF hand-S2-S3 loop interface. Our simulations unveiled two key interactions: (1) K4101 (EF hand) with D4730 (S2-S3 loop) and (2) E4075, Q4078, and D4079 (EF hand) with R4736 (S2-S3 loop). To probe the functional significance of these interactions, we constructed mutant RyR1 complementary DNAs and expressed them in HEK293 cells for [3H]ryanodine binding assays. Our results demonstrated that mutations in the EF hand, specifically K4101E and K4101M, resulted in reduced affinities for Ca2+/Mg2+-dependent inhibitions. Interestingly, the K4101E mutation increased the affinity for Ca2+-dependent activation. Conversely, mutations in the S2-S3 loop, D4730K and D4730N, did not significantly change the affinities for Ca2+/Mg2+-dependent inhibitions. Our previous finding that skeletal disease-associated RyR1 mutations, R4736Q and R4736W, impaired Ca2+-dependent inhibition, is consistent with the current results. In silico mutagenesis analysis aligned with our functional data, indicating altered hydrogen bonding patterns upon mutations. Taken together, our findings emphasize the critical role of the EF hand-S2-S3 loop interaction in Ca2+/Mg2+-dependent inhibition of RyR1 and provide insights into potential therapeutic strategies targeting this domain interaction for the treatment of skeletal myopathies.

Mapping co-regulatory interactions among ligand-binding sites in ryanodine receptor 1.

Ryanodine receptor 1 (RyR1) is an intracellular calcium ion (Ca2+ ) release channel required for skeletal muscle contraction. Although cryo-electron microscopy identified binding sites of three coactivators Ca2+ , ATP, and caffeine (CFF), the mechanism of co-regulation and synergy of these activators is unknown. Here, we report allosteric connections among the three ligand-binding sites and pore region in (i) Ca2+ bound-closed, (ii) ATP/CFF bound-closed, (iii) Ca2+ /ATP/CFF bound-closed, and (iv) Ca2+ /ATP/CFF bound-open RyR1 states. We identified two dominant networks of interactions that mediate communication between the Ca2+ -binding site and pore region in Ca2+ bound-closed state, which partially overlapped with the pore communications in ATP/CFF bound-closed RyR1 state. In Ca2+ /ATP/CFF bound-closed and -open RyR1 states, co-regulatory interactions were analogous to communications in the Ca2+ bound-closed and ATP/CFF bound-closed states. Both ATP- and CFF-binding sites mediate communication between the Ca2+ -binding site and the pore region in Ca2+ /ATP/CFF bound-open RyR1 structure. We conclude that Ca2+ , ATP, and CFF propagate their effects to the pore region through a network of overlapping interactions that mediate allosteric control and molecular synergy in channel regulation.

Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1).

Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+. We report that either phosphomethylphosphonic acid adenylate ester (AMPPCP), a nonhydrolyzable ATP analog, or Caf can activate RyR1 in the absence or the presence of Ca2+. However, enhanced RyR1 activation occurred in the presence of Ca2+, AMPPCP, and Caf. In the absence of Ca2+, Na+ inhibited [3H]ryanodine binding without impairing RyR1 activation by AMPPCP and Caf. Computational analysis suggested that Ca2+-, ATP-, and Caf-binding sites modulate RyR1 protein stability through interactions with the carboxyterminal domain and other domains in the activation core. In the presence of ATP and Caf but the absence of Ca2+, Na+ is predicted to inhibit RyR1 by interacting with the Ca2+-binding site. Our data suggested that ATP and Caf binding affected the conformation of the Ca2+-binding site, and conversely, Ca2+ binding affected the conformation of the ATP- and Caf-binding sites. We conclude that Ca2+, ATP, and Caf regulate RyR1 through a network of allosteric interactions involving the Ca2+-, ATP-, and Caf-binding sites.

Single-channel properties of skeletal muscle ryanodine receptor pore Δ4923FF4924 in two brothers with a lethal form of fetal akinesia.

Ryanodine receptor ion channels (RyR1s) release Ca2+ ions from the sarcoplasmic reticulum to regulate skeletal muscle contraction. By whole-exome sequencing, we identified the heterozygous RYR1 variant c.14767_14772del resulting in the in-frame deletion p.(Phe4923_Phe4924del) in two brothers with a lethal form of the fetal akinesia deformation syndrome (FADS). The two deleted phenylalanines (RyR1-Δ4923FF4924) are located in the S6 pore-lining helix of RyR1. Clinical features in one of the two siblings included severe hypotonia, thin ribs, swallowing inability, and respiratory insufficiency that caused early death. Functional consequences of the RyR1-Δ4923FF4924 variant were determined using recombinant 2,200-kDa homotetrameric and heterotetrameric RyR1 channel complexes that were expressed in HEK293 cells and characterized by cellular, electrophysiological, and computational methods. Cellular Ca2+ release in response to caffeine indicated that the homotetrameric variant formed caffeine-sensitive Ca2+ conducting channels in HEK293 cells. In contrast, the homotetrameric channel complex was not activated by Ca2+ and did not conduct Ca2+ based on single-channel measurements. The computational analysis suggested decreased protein stability and loss of salt bridge interactions between RyR1-R4944 and RyR1-D4938, increasing the electrostatic interaction energy of Ca2+ in a region 20 Å from the mutant site. Co-expression of wild-type and mutant RyR1s resulted in Ca2+-dependent channel activities that displayed intermediate Ca2+ conductances and suggested maintenance of a reduced Ca2+ release in the two patients. Our findings reveal that the RYR1 pore variant p.(Phe4923_Phe4924del) attenuates the flow of Ca2+ through heterotetrameric channels, but alone was not sufficient to cause FADS, indicating additional genetic factors to be involved.

A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

Ca2+-mediated activation of the skeletal-muscle ryanodine receptor ion channel.

Cryo-electron micrograph studies recently have identified a Ca2+-binding site in the 2,200-kDa ryanodine receptor ion channel (RyR1) in skeletal muscle. To clarify the role of this site in regulating RyR1 activity, here we applied mutational, electrophysiological, and computational methods. Three amino acid residues that interact directly with Ca2+ were replaced, and these RyR1 variants were expressed in HEK293 cells. Single-site RyR1-E3893Q, -E3893V, -E3967Q, -E3967V, and -T5001A variants and double-site RyR1-E3893Q/E3967Q and -E3893V/E3967V variants displayed cellular Ca2+ release in response to caffeine, which indicated that they retained functionality as caffeine-sensitive, Ca2+-conducting channels in the HEK293 cell system. Using [3H]ryanodine binding and single-channel measurements of membrane isolates, we found that single- and double-site RyR1-E3893 and -E3967 variants are not activated by Ca2+ We also noted that RyR1-E3893Q/E3967Q and -E3893V/E3967V variants maintain caffeine- and ATP-induced activation and that RyR1-E3893Q/E3967Q is inhibited by Mg2+ and elevated Ca2+ RyR1-T5001A exhibited decreased Ca2+ sensitivity compared with WT-RyR1 in single-channel measurements. Computational methods suggested that electrostatic interactions between Ca2+ and negatively charged glutamate residues have a critical role in transducing the functional effects of Ca2+ on RyR1. We conclude that the removal of negative charges in the recently identified RyR1 Ca2+-binding site impairs RyR1 activation by physiological Ca2+ concentrations and results in loss of binding to Ca2+ or reduced Ca2+ affinity of the binding site.

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2.

The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca2+ release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca2+ in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K+ conductance and reduced selectivity for Ca2+ compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca2+ concentrations and nearly fully opened at 2 µm cytosolic or sarcoplasmic reticulum luminal Ca2+, and Ca2+- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca2+ at low cytosolic Ca2+ levels. A model suggested that luminal Ca2+ activates RyR1 by accessing a recently identified cytosolic Ca2+-binding site in the open channel as the Ca2+ ions pass through the pore.

The structural basis of ryanodine receptor ion channel function.

Large-conductance Ca2+ release channels known as ryanodine receptors (RyRs) mediate the release of Ca2+ from an intracellular membrane compartment, the endo/sarcoplasmic reticulum. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four ∼12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases. RyRs share ∼70% sequence identity, with the greatest sequence similarity in the C-terminal region that forms the transmembrane, ion-conducting domain comprising ∼500 amino acids. The remaining ∼4,500 amino acids form the large regulatory cytoplasmic "foot" structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine. Recent work describing the near atomic structures of mammalian skeletal and cardiac muscle RyRs provides a structural basis for the regulation of the RyRs by their multiple effectors.

Two EF-hand motifs in ryanodine receptor calcium release channels contribute to isoform-specific regulation by calmodulin.

The mammalian ryanodine receptor Ca2+ release channel (RyR) has a single conserved high affinity calmodulin (CaM) binding domain. However, the skeletal muscle RyR1 is activated and cardiac muscle RyR2 is inhibited by CaM at submicromolar Ca2+. This suggests isoform-specific domains are involved in RyR regulation by CaM. To gain insight into the differential regulation of cardiac and skeletal muscle RyRs by CaM, RyR1/RyR2 chimeras and mutants were expressed in HEK293 cells, and their single channel activities were measured using a lipid bilayer method. All RyR1/RyR2 chimeras and mutants were inhibited by CaM at 2µM Ca2+, consistent with CaM inhibition of RyR1 and RyR2 at micromolar Ca2+ concentrations. An RyR1/RyR2 chimera with RyR1 N-terminal amino acid residues (aa) 1-3725 and RyR2 C-terminal aa 3692-4968 were inhibited by CaM at <1µM Ca2+ similar to RyR2. In contrast, RyR1/RyR2 chimera with RyR1 aa 1-4301 and RyR2 4254-4968 was activated at <1µM Ca2+ similar to RyR1. Replacement of RyR1 aa 3726-4298 with corresponding residues from RyR2 conferred CaM inhibition at <1µM Ca2+, which suggests RyR1 aa 3726-4298 are required for activation by CaM. Characterization of additional RyR1/RyR2 chimeras and mutants in two predicted Ca2+ binding motifs in RyR1 aa 4081-4092 (EF1) and aa 4116-4127 (EF2) suggests that both EF-hand motifs and additional sequences in the large N-terminal regions are required for isoform-specific RyR1 and RyR2 regulation by CaM at submicromolar Ca2+ concentrations.

Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor.

The type 1 ryanodine receptor (RyR1) mediates Ca2+ release from the sarcoplasmic reticulum to initiate skeletal muscle contraction and is associated with muscle diseases, malignant hyperthermia, and central core disease. To better understand RyR1 channel function, we investigated the molecular mechanisms of channel gating and ion permeation. An adequate model of channel gating requires accurate, high-resolution models of both open and closed states of the channel. To this end, we generated an open-channel RyR1 model using molecular simulations to pull Ca2+ through the pore constriction site of a closed-channel RyR1 structure determined at 3.8-Šresolution. Importantly, we find that our open-channel model is consistent with the RyR1 and cardiac RyR (RyR2) open-channel structures reported while this paper was in preparation. Both our model and the published structures show similar rotation of the upper portion of the pore-lining S6 helix away from the 4-fold channel axis and twisting of Ile-4937 at the channel constriction site out of the channel pore. These motions result in a minimum open-channel pore radius of ∼3 Šformed by Gln-4933, rather than Ile-4937 in the closed-channel structure. We also present functional support for our model by mutations around the closed- and open-channel constriction sites (Gln-4933 and Ile-4937). Our results indicate that use of ion-pulling simulations produces a RyR1 open-channel model, which can provide insights into the mechanisms of channel opening complementing those from the structural data.

mTOR signaling in mice with dysfunctional cardiac ryanodine receptor ion channel.

Simultaneous substitution of three amino acid residues in the calmodulin binding domain (W3587A/L3591D/F3603A, ADA) of the cardiac ryanodine receptor ion channel (RyR2) impairs calmodulin inhibition of RyR2 and causes cardiac hypertrophy and early death of Ryr2ADA/ADA mice. To determine the physiological significance of growth promoting signaling molecules, the protein and phosphorylation levels of Ser/Thr kinase mTOR and upstream and downstream signaling molecules were determined in hearts of wild-type and Ryr2ADA/ADA mice. Phosphorylation of mTOR at Ser-2448, and mTOR downstream targets p70S6 kinase at Thr-389, S6 ribosomal protein at Ser-240/244, and 4E-BP1 at Ser-65 were increased. However, there was no increased phosphorylation of mTOR upstream kinases PDK1 at Ser-241, AKT at Thr-308, AMPK at Thr-172, and ERK1/2 at Thr-202/Tyr204. To confirm a role for mTOR signaling in the development of cardiac hypertrophy, rapamycin, an inhibitor of mTOR, was injected into wild-type and mutant mice. Rapamycin decreased mouse heart-to-body weight ratio, improved cardiac performance, and decreased phosphorylation of mTOR and downstream targets p70S6K and S6 in 10-day-old Ryr2ADA/ADA mice but did not extend longevity. Taken together, the results link a dysfunctional RyR2 to an altered activity of signaling molecules that regulate cardiac growth and function.

PHD2/3-dependent hydroxylation tunes cardiac response to ß-adrenergic stress via phospholamban.

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic ß-adrenergic stress-induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.

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

Type 1 ryanodine receptors (RyR1s) release Ca(2+) 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 Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]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 Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) 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.

Dyad content is reduced in cardiac myocytes of mice with impaired calmodulin regulation of RyR2.

In cardiac muscle, calmodulin (CaM) regulates the activity of several membrane proteins involved in Ca(2+) homeostasis (CaV1.2; RyR2, SERCA2, PMCA). Three engineered amino acid substitutions in the CaM binding site of the cardiac ryanodine receptor (RyR2) in mice (Ryr2 (ADA/ADA) ) strongly affect cardiac function, with impaired CaM inhibition of RyR2, reduced SR Ca(2+) sequestration, and early cardiac hypertrophy and death (Yamaguchi et al., J Clin Invest 117:1344-1353, 2007). We have examined the ultrastructure and RyR2 immunolocalization in WT and Ryr2 (ADA/ADA) hearts at ~10 days after birth. The myocytes show only minor evidence of structural damage: some increase in intermyofibrillar space, with occasional areas of irregular SR disposition and an increase in frequency of smaller myofibrils, despite an increase of about 15 % in average myocyte cross sectional area. Z line streaming, a sign of myofibrillar stress, is limited and fairly rare. Immunolabeling with an anti-RyR2 antibody shows that RyR-positive foci located at the level of the Z lines are less frequent in mutant hearts. A dramatic decrease in the frequency and size of dyads, accompanied by a decrease in occupancy of the gap by RyR2, but without obvious alterations in location and general structure is a notable ultrastructural feature. The data suggest that the uneven distribution of dyads or calcium release sites within the cells resulting from an overall reduction in RyR2 content may contribute to the poor cardiac performance and early death of Ryr2 (ADA/ADA) mice. An unusual fragmentation of mitochondria, perhaps related to imbalances in free cytoplasmic calcium levels, accompanies these changes.

IL-6/STAT3 signaling in mice with dysfunctional type-2 ryanodine receptor.

Mice with genetically modified cardiac ryanodine receptor (Ryr2 (ADA/ADA) mice) are impaired in regulation by calmodulin, develop severe cardiac hypertrophy and die about 2 weeks after birth. We hypothesized that the interleukin 6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling pathway has a role in the development of the Ryr2 (ADA/ADA) cardiac hypertrophy phenotype, and determined cardiac function and protein levels of IL-6, phosphorylation levels of STAT3, and downstream targets c-Fos and c-Myc in wild-type and RyR2 (ADA/ADA) mice, mice with a disrupted IL-6 gene, and mice treated with STAT3 inhibitor NSC74859. IL-6 protein levels were increased at postnatal day 1 but not day 10, whereas pSTAT3-Tyr705/STAT3 ratio and c-Fos and c-Myc protein levels increased in hearts of 10-day but not 1-day old Ryr2 (ADA/ADA) mice compared with wild type. Both STAT3 and pSTAT3-Tyr705 accumulated in the nuclear fraction of 10-day old Ryr2 (ADA/ADA) mice compared with wild type. Ryr2 (ADA /ADA) /IL-6(-/-) mice lived 1.5 times longer, had decreased heart to body weight ratio, and reduced c-Fos and c-Myc protein levels. The STAT3 inhibitor NSC74859 prolonged life span by 1.3-fold, decreased heart to body weight ratio, increased cardiac performance, and decreased pSTAT-Tyr705/STAT3 ratio and IL-6, c-Fos and c-Myc protein levels of Ryr2 (ADA /ADA) mice. The results suggest that upregulation of IL-6 and STAT3 signaling contributes to cardiac hypertrophy and early death of mice with a dysfunctional ryanodine receptor. They further suggest that STAT3 inhibitors may be clinically useful agents in patients with altered Ca(2+) handling in the heart.

Selecting ions by size in a calcium channel: the ryanodine receptor case study.

Many calcium channels can distinguish between ions of the same charge but different size. For example, when cations are in direct competition with each other, the ryanodine receptor (RyR) calcium channel preferentially conducts smaller cations such as Li(+) and Na(+) over larger ones such as K(+) and Cs(+). Here, we analyze the physical basis for this preference using a previously established model of RyR permeation and selectivity. Like other calcium channels, RyR has four aspartate residues in its GGGIGDE selectivity filter. These aspartates have their terminal carboxyl group in the pore lumen, which take up much of the available space for permeating ions. We find that small ions are preferred by RyR because they can fit into this crowded environment more easily.

Inhibition of CaMKII does not attenuate cardiac hypertrophy in mice with dysfunctional ryanodine receptor.

In cardiac muscle, the release of calcium ions from the sarcoplasmic reticulum through ryanodine receptor ion channels (RyR2s) leads to muscle contraction. RyR2 is negatively regulated by calmodulin (CaM) and by phosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII). Substitution of three amino acid residues in the CaM binding domain of RyR2 (RyR2-W3587A/L3591D/F3603A, RyR2ADA) impairs inhibition of RyR2 by CaM and results in cardiac hypertrophy and early death of mice carrying the RyR2ADA mutation. To test the cellular function of CaMKII in cardiac hypertrophy, mutant mice were crossed with mice expressing the CaMKII inhibitory AC3-I peptide or the control AC3-C peptide in the myocardium. Inhibition of CaMKII by AC3-I modestly reduced CaMKII-dependent phosphorylation of RyR2 at Ser-2815 and markedly reduced CaMKII-dependent phosphorylation of SERCA2a regulatory subunit phospholamban at Thr-17. However the average life span and heart-to-body weight ratio of Ryr2ADA/ADA mice expressing the inhibitory peptide were not altered compared to control mice. In Ryr2ADA/ADA homozygous mice, AC3-I did not alter cardiac morphology, enhance cardiac function, improve sarcoplasmic reticulum Ca2+ handling, or suppress the expression of genes implicated in cardiac remodeling. The results suggest that CaMKII was not required for the rapid development of cardiac hypertrophy in Ryr2ADA/ADA mice.

Pore dynamics and conductance of RyR1 transmembrane domain.

Ryanodine receptors (RyR) are calcium release channels, playing a major role in the regulation of muscular contraction. Mutations in skeletal muscle RyR (RyR1) are associated with congenital diseases such as malignant hyperthermia and central core disease (CCD). The absence of high-resolution structures of RyR1 has limited our understanding of channel function and disease mechanisms at the molecular level. Previously, we have reported a hypothetical structure of the RyR1 pore-forming region, obtained by homology modeling and supported by mutational scans, electrophysiological measurements, and cryo-electron microscopy. Here, we utilize the expanded model encompassing six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the proposed pore structure can sustain ion currents measured in single-channel experiments. We observe a stable pore structure on timescales of 0.2 µs, with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. We further suggest that stability of the selectivity filter critically depends on the interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of polar substitution I4897T results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca(2+) conductance.

Cardiac myocyte Z-line calmodulin is mainly RyR2-bound, and reduction is arrhythmogenic and occurs in heart failure.

RATIONALE: Calmodulin (CaM) associates with cardiac ryanodine receptor type-2 (RyR2) as an important regulator. Defective CaM-RyR2 interaction may occur in heart failure, cardiac hypertrophy, and catecholaminergic polymorphic ventricular tachycardia. However, the in situ binding properties for CaM-RyR2 are unknown. OBJECTIVE: We sought to measure the in situ binding affinity and kinetics for CaM-RyR2 in normal and heart failure ventricular myocytes, estimate the percentage of Z-line-localized CaM that is RyR2-bound, and test cellular function of defective CaM-RyR2 interaction. METHODS AND RESULTS: Using fluorescence resonance energy transfer in permeabilized myocytes, we specifically resolved RyR2-bound CaM from other potential binding targets and measured CaM-RyR2 binding affinity in situ (Kd=10-20 nmol/L). Using RyR2(ADA/+) knock-in mice, in which half of the CaM-RyR2 binding is suppressed, we estimated that >90% of Z-line CaM is RyR2-bound. Functional tests indicated a higher propensity for Ca2+ wave production and stress-induced ventricular arrhythmia in RyR2(ADA/+) mice. In a post-myocardial infarction rat heart failure model, we detected a decrease in the CaM-RyR2 binding affinity (Kd≈51 nmol/L; ≈3-fold increase) and unaltered RyR2 affinity for the FK506-binding protein FKBP12.6 (Kd~0.8 nmol/L). CONCLUSIONS: CaM binds to RyR2 with high affinity in cardiac myocytes. Physiologically, CaM is bound to >70% of RyR2 monomers and inhibits sarcoplasmic reticulum Ca2+ release. RyR2 is the major binding site for CaM along the Z-line in cardiomyocytes, and dissociating CaM from RyR2 can cause severe ventricular arrhythmia. In heart failure, RyR2 shows decreased CaM affinity, but unaltered FKBP 12.6 affinity.

Cardiac calcium signalling pathologies associated with defective calmodulin regulation of type 2 ryanodine receptor.

Cardiac ryanodine receptor (RyR2) is a homotetramer of 560 kDa polypeptides regulated by calmodulin (CaM), which decreases its open probability at diastolic and systolic Ca(2+) concentrations. Point mutations in the CaM-binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2(ADA)) in mice result in severe cardiac hypertrophy, poor left ventricle contraction and death by postnatal day 16, suggesting that CaM inhibition of RyR2 is required for normal cardiac function. Here, we report on Ca(2+) signalling properties of enzymatically isolated, Fluo-4 dialysed whole cell clamped cardiac myocytes from 10-15-day-old wild-type (WT) and homozygous Ryr2(ADA/ADA) mice. Spontaneously occurring Ca(2+) spark frequency, measured at -80 mV, was 14-fold lower in mutant compared to WT myocytes. ICa, though significantly smaller in mutant myocytes, triggered Ca(2+) transients that were of comparable size to those of WT myocytes, but with slower activation and decay kinetics. Caffeine-triggered Ca(2+) transients were about three times larger in mutant myocytes, generating three- to four-fold bigger Na(+)-Ca(2+) exchanger NCX currents (INCX). Mutant myocytes often exhibited Ca(2+) transients of variable size and duration that were accompanied by similarly alternating and slowly activating INCX. The data suggest that RyR2(ADA) mutation produces significant reduction in ICa density and ICa-triggered Ca(2+) release gain, longer but infrequently occurring Ca(2+) sparks, larger sarcoplasmic reticulum Ca(2+) loads, and spontaneous Ca(2+) releases accompanied by activation of large and potentially arrhythmogenic inward INCX.