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1.
Nat Commun ; 12(1): 807, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547325

RESUMO

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological 'intermediate' conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.


Assuntos
Apoproteínas/química , Cálcio/química , Calmodulina/química , Hipertermia Maligna/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Substituição de Aminoácidos , Animais , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/química , Arginina/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Cisteína/metabolismo , Expressão Gênica , Humanos , Transporte de Íons , Hipertermia Maligna/genética , Hipertermia Maligna/patologia , Modelos Moleculares , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Suínos
2.
Nat Commun ; 11(1): 4734, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948759

RESUMO

A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged in the binding of ligands. The functional motions differ substantially from those inferred from static structures in the nature of conformationally active structural domains, the sequence and extent of conformational motions, and the way allosteric signals are transduced within and between domains. Our approach highlights the importance of combining experiment, advanced data analysis, and molecular simulations.


Assuntos
Agonistas dos Canais de Cálcio/química , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sítios de Ligação , Microscopia Crioeletrônica , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
3.
Nat Commun ; 11(1): 3709, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709843

RESUMO

Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.


Assuntos
Canais de Cálcio/química , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Razão Sinal-Ruído , Imagem Individual de Molécula , Fluxo de Trabalho
4.
Nat Commun ; 11(1): 922, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066742

RESUMO

Ryanodine receptors (RyR) are ion channels responsible for the release of Ca2+ from the sarco/endoplasmic reticulum and play a crucial role in the precise control of Ca2+ concentration in the cytosol. The detailed permeation mechanism of Ca2+ through RyR is still elusive. By using molecular dynamics simulations with a specially designed Ca2+ model, we show that multiple Ca2+ ions accumulate in the upper selectivity filter of RyR1, but only one Ca2+ can occupy and translocate in the narrow pore at a time, assisted by electrostatic repulsion from the Ca2+ within the upper selectivity filter. The Ca2+ is nearly fully hydrated with the first solvation shell intact during the whole permeation process. These results suggest a remote knock-on permeation mechanism and one-at-a-time occupation pattern for the hydrated Ca2+ within the narrow pore, uncovering the basis underlying the high permeability and low selectivity of the RyR channels.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Simulação de Dinâmica Molecular , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/química , Calpaína/metabolismo , Cátions Bivalentes/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Eletricidade Estática , Relação Estrutura-Atividade
5.
J Agric Food Chem ; 68(6): 1731-1740, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31951399

RESUMO

Diamide insecticides targeting ryanodine receptors (RyRs) are a major class of pesticides used to control a wide range of agricultural pests, but their efficacies have been reduced dramatically by the recent emergence of resistance mutations. There is a pressing need to develop novel insecticides, targeting distinct and novel binding sites within insect RyRs to overcome the resistance crisis; however, the limited structural information on insect RyRs is a major roadblock to our understanding of their molecular mechanisms. Here, we report the crystal structure of the RyR SPRY2 domain from the diamondback moth (DBM), Plutella xylostella, a destructive agricultural pest worldwide that has developed resistance to all classes of insecticide at 2.06 Å resolution. The overall fold of DBM SPRY2 is similar to its mammalian homolog, but it shows distinct conformations in several loops. Docking it into the recently published cryo-electron microscope structure of the full-length RyR reveals that two insect-specific loops interact with the BSol domain from the neighboring subunit. The SPRY2-BSol interface will change the conformation upon channel gating, indicating that it might be a potential targeting site for insect-specific insecticides. Interestingly, several previously identified disease-causing mutations also lie in the same interface, implying that this interface is important for channel gating. Another insect-specific loop located in the SPRY2-SPRY3 interface might indirectly affect another gating interface between SPRY3 and Repeat34.


Assuntos
Diamida/química , Proteínas de Insetos/química , Inseticidas/química , Mariposas/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Diamida/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Simulação de Acoplamento Molecular , Mariposas/química , Mariposas/efeitos dos fármacos , Mariposas/genética , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
6.
Adv Exp Med Biol ; 1131: 321-336, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646516

RESUMO

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca2+ storage compartment, and release Ca2+ during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR. RyR1 and RyR2 are the major RyR isoforms in skeletal and cardiac muscle, respectively, and RyR3 is expressed in various tissues along with the other two isoforms. A prominent feature of RyRs is that the Ca2+ release channel activities of RyRs are regulated by calcium ions; therefore, intracellular Ca2+ release controls positive- and negative-feedback phenomena through the RyRs. RyR channel activities are also regulated by Ca2+ indirectly, i.e. through Ca2+ binding proteins at both cytosolic and sarco/endoplasmic reticulum luminal sides. Here, I summarize Ca2+-dependent feedback regulation of RyRs including recent progress in the structure/function aspects.


Assuntos
Cálcio , Regulação da Expressão Gênica , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Citosol/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Relação Estrutura-Atividade
7.
Circ Res ; 126(4): 417-435, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31805819

RESUMO

RATIONALE: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. OBJECTIVE: In cardiomyocytes, spontaneous Ca2+ waves, triggered by store overload-induced Ca2+ release mediated by the RyR2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR2-mediated Ca2+ release machinery that directly modulates store overload-induced Ca2+ release activity via CTT. METHODS AND RESULTS: We show that cardiomyocytes derived from the TRIC-A-/- (TRIC-A knockout) mice display dysregulated Ca2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR2. Modeling and docking studies reveal potential sites on RyR2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR2, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca2+ oscillations. Ca2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca2+ leak across the endoplasmic reticulum by directly targeting RyR2 to modulate store overload-induced Ca2+ release. Moreover, synthetic CTT-A peptide facilitates RyR2 activity in lipid bilayer reconstitution system, enhances Ca2+ sparks in permeabilized TRIC-A-/- cardiomyocytes, and induces intracellular Ca2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A-/- mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. CONCLUSIONS: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR2 to modulate sarcoplasmic reticulum Ca2+ handling in cardiac muscle.


Assuntos
Cálcio/metabolismo , Canais Iônicos/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Sinalização do Cálcio , Cardiotônicos/farmacologia , Eletrocardiografia/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Fibrose/genética , Fibrose/fisiopatologia , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Isoproterenol/farmacologia , Camundongos Knockout , Simulação de Acoplamento Molecular , Miocárdio/citologia , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/metabolismo
8.
Proc Natl Acad Sci U S A ; 116(51): 25575-25582, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31792195

RESUMO

The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5'-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.


Assuntos
Regulação Alostérica/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Microscopia Crioeletrônica , Modelos Moleculares , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Suínos
9.
BMC Biol ; 17(1): 77, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31597572

RESUMO

BACKGROUND: Ryanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation. RESULTS: Here we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis. CONCLUSIONS: The crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.


Assuntos
Proteínas de Insetos/química , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Clonagem Molecular , Escherichia coli/genética , Lepidópteros/metabolismo , Fosforilação , Conformação Proteica
10.
Cell Calcium ; 82: 102063, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31401388

RESUMO

Here we report the structure of the widely utilized calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitor KN93 bound to the Ca2+-sensing protein CaM. KN93 is widely believed to inhibit CaMKII by binding to the kinase. The CaM-KN93 interaction is significant as it can interfere with the interaction between CaM and it's physiological targets, thereby raising the possibility of ascribing modified protein function to CaMKII phosphorylation while concealing a CaM-protein interaction. NMR spectroscopy, stopped-flow kinetic measurements, and x-ray crystallography were used to characterize the structure and biophysical properties of the CaM-KN93 interaction. We then investigated the functional properties of the cardiac Na+ channel (NaV1.5) and ryanodine receptor (RyR2). We find that KN93 disrupts a high affinity CaM-NaV1.5 interaction and alters channel function independent of CaMKII. Moreover, KN93 increases RyR2 Ca2+ release in cardiomyocytes independent of CaMKII. Therefore, when interpreting KN93 data, targets other than CaMKII need to be considered.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Benzilaminas/farmacologia , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Calmodulina/química , Calmodulina/genética , Células Cultivadas , Cristalografia por Raios X , Humanos , Miócitos Cardíacos , Canal de Sódio Disparado por Voltagem NAV1.5/química , Fosforilação , Ligação Proteica , Conformação Proteica , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Sulfonamidas/farmacologia
11.
Mol Pharmacol ; 96(3): 401-407, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31337666

RESUMO

Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca2+ release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg2+ was demonstrated to be the essential factor for dantrolene to inhibit Ca2+ release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca2+ release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca2+ release experiments demonstrated that the effect of dantrolene and Mg2+ was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 µM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg2+ and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg2+-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.


Assuntos
Trifosfato de Adenosina/metabolismo , Dantroleno/farmacologia , Magnésio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Sítios de Ligação , Cálcio/metabolismo , Dantroleno/química , Masculino , Modelos Moleculares , Conformação Molecular , Músculo Esquelético/metabolismo , Ligação Proteica , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química
12.
Nature ; 572(7769): 347-351, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31278385

RESUMO

The high-conductance intracellular calcium (Ca2+) channel RyR2 is essential for the coupling of excitation and contraction in cardiac muscle. Among various modulators, calmodulin (CaM) regulates RyR2 in a Ca2+-dependent manner. Here we reveal the regulatory mechanism by which porcine RyR2 is modulated by human CaM through the structural determination of RyR2 under eight conditions. Apo-CaM and Ca2+-CaM bind to distinct but overlapping sites in an elongated cleft formed by the handle, helical and central domains. The shift in CaM-binding sites on RyR2 is controlled by Ca2+ binding to CaM, rather than to RyR2. Ca2+-CaM induces rotations and intradomain shifts of individual central domains, resulting in pore closure of the PCB95 and Ca2+-activated channel. By contrast, the pore of the ATP, caffeine and Ca2+-activated channel remains open in the presence of Ca2+-CaM, which suggests that Ca2+-CaM is one of the many competing modulators of RyR2 gating.


Assuntos
Calmodulina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoproteínas/metabolismo , Sítios de Ligação , Cafeína/metabolismo , Cálcio/metabolismo , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Reprodutibilidade dos Testes , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Suínos
13.
FEBS J ; 286(22): 4554-4578, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31230402

RESUMO

A number of calmodulin (CaM) mutations cause severe cardiac arrhythmias, but their arrhythmogenic mechanisms are unclear. While some of the arrhythmogenic CaM mutations have been shown to impair CaM-dependent inhibition of intracellular Ca2+ release through the ryanodine receptor type 2 (RyR2), the impact of a majority of these mutations on RyR2 function is unknown. Here, we investigated the effect of 14 arrhythmogenic CaM mutations on the CaM-dependent RyR2 inhibition. We found that all the arrhythmogenic CaM mutations tested diminished CaM-dependent inhibition of RyR2-mediated Ca2+ release and increased store-overload induced Ca2+ release (SOICR) in HEK293 cells. Moreover, all the arrhythmogenic CaM mutations tested either failed to inhibit or even promoted RyR2-mediated Ca2+ release in permeabilized HEK293 cells with elevated cytosolic Ca2+ , which was markedly different from the inhibitory action of CaM wild-type. The CaM mutations also altered the Ca2+ -dependency of CaM binding to the RyR2 CaM-binding domain. These results demonstrate that diminished inhibition, and even facilitated activation, of RyR2-mediated Ca2+ release is a common defect of arrhythmogenic CaM mutations.


Assuntos
Arritmias Cardíacas/genética , Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/genética , Mutação , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sítios de Ligação , Canais de Cálcio Tipo L/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
14.
JCI Insight ; 4(11)2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31167968

RESUMO

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by a single point mutation in the cardiac type 2 ryanodine receptor (RyR2). Using a knockin (KI) mouse model (R2474S/+), we previously reported that a single point mutation within the RyR2 sensitizes the channel to agonists, primarily mediated by defective interdomain interaction within the RyR2 and subsequent dissociation of calmodulin (CaM) from the RyR2. Here, we examined whether CPVT can be genetically rescued by enhancing the binding affinity of CaM to the RyR2. We first determined whether there is a possible amino acid substitution within the CaM-binding domain in the RyR2 (3584-3603 residues) that can enhance its binding affinity to CaM and found that V3599K substitution showed the highest binding affinity of CaM to the CaM-binding domain. Hence, we generated a heterozygous KI mouse model (V3599K/+) with a single amino acid substitution in the CaM-binding domain of the RyR2 and crossbred it with the heterozygous CPVT-associated R2474S/+-KI mouse to obtain a double-heterozygous R2474S/V3599K-KI mouse model. The CPVT phenotypes - bidirectional or polymorphic ventricular tachycardia, spontaneous Ca2+ transients, and Ca2+ sparks - were all inhibited in the R2474S/V3599K mice. Thus, enhancement of the CaM-binding affinity of the RyR2 is essential to prevent CPVT-associated arrhythmogenesis.


Assuntos
Calmodulina , Canal de Liberação de Cálcio do Receptor de Rianodina , Taquicardia Ventricular , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miócitos Cardíacos , Ligação Proteica/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
15.
Am J Physiol Cell Physiol ; 317(2): C358-C365, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31166712

RESUMO

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.


Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico , Músculo Esquelético/metabolismo , Mutação de Sentido Incorreto , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sítios de Ligação , Cafeína/farmacologia , Agonistas dos Canais de Cálcio/farmacologia , Células HEK293 , Humanos , Potenciais da Membrana , Músculo Esquelético/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Relação Estrutura-Atividade
16.
J Pharmacol Exp Ther ; 371(1): 219-230, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31209099

RESUMO

Vesicular monoamine transporter-2 is expressed in the presynaptic secretory vesicles membrane in the brain. Its blockade by tetrabenazine (TBZ) causes depletion of dopamine at striatal basal ganglia; this is the mechanism underlying its long-standing use in the treatment of Huntington's disease. In the frame of a project aimed at investigating the kinetics of exocytosis from vesicles with partial emptying of their neurotransmitter, we unexpectedly found that TBZ facilitates exocytosis; thus, we decided to characterize such effect. We used bovine chromaffin cells (BCCs) challenged with repeated pulses of high K+ Upon repeated K+ pulsing, the exocytotic catecholamine release responses were gradually decaying. However, when cells were exposed to TBZ, responses were mildly augmented and decay rate delayed. Facilitation of exocytosis was not due to Ca2+ entry blockade through voltage-activated calcium channels (VACCs) because, in fact, TBZ mildly blocked the whole-cell Ca2+ current. However, TBZ mimicked the facilitatory effects of exocytosis elicited by BayK8644 (L-subtype VACC agonist), an effect blocked by nifedipine (VACC antagonist). On the basis that TBZ augmented the secretory responses to caffeine (but not those of histamine), we monitored its effects on cytosolic Ca2+ elevations ([Ca2+]c) triggered by caffeine or histamine. While the responses to caffeine were augmented twice by TBZ, those of histamine were unaffected; the same happened in rat cortical neurons. Hence, we hypothesize that TBZ facilitates exocytosis by increasing Ca2+ release through the endoplasmic reticulum ryanodine receptor channel (RyR). Confirming this hypothesis are docking results, showing an interaction of TBZ with RyRs. This is consonant with the existence of a healthy Ca2+-induced-Ca2+-release mechanism in BCCs. SIGNIFICANCE STATEMENT: A novel mechanism of action for tetrabenazine (TBZ), a drug used in the therapy of Huntington's disease (HD), is described here. Such mechanism consists of facilitation by combining TBZ with the ryanodine receptor of the endoplasmic reticulum, thereby increasing Ca2+-induced Ca2+ release. This novel mechanism should be taken into account when considering the efficacy and/or safety of TBZ in the treatment of chorea associated with HD and other disorders. Additionally, it could be of interest in the development of novel medicines to treat these pathological conditions.


Assuntos
Inibidores da Captação Adrenérgica/farmacologia , Sinalização do Cálcio , Células Cromafins/efeitos dos fármacos , Exocitose , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Tetrabenazina/farmacologia , Animais , Sítios de Ligação , Canais de Cálcio Tipo N/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/química
17.
Mol Cell ; 75(1): 39-52.e4, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31078384

RESUMO

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels controlling essential cellular functions. RyRs are targeted by cyclic AMP (cAMP)-dependent protein kinase A (PKA), a controversial regulation implicated in disorders ranging from heart failure to Alzheimer's. Using crystal structures, we show that the phosphorylation hotspot domain of RyR2 embraces the PKA catalytic subunit, with an extensive interface not seen in PKA complexes with peptides. We trapped an intermediary open-form PKA bound to the RyR2 domain and an ATP analog, showing that PKA can engage substrates in an open form. Phosphomimetics or prior phosphorylation at nearby sites in RyR2 either enhance or reduce the activity of PKA. Finally, we show that a phosphomimetic at S2813, a well-known target site for calmodulin-dependent kinase II, induces the formation of an alpha helix in the phosphorylation domain, resulting in increased interactions and PKA activity. This shows that the different phosphorylation sites in RyR2 are not independent.


Assuntos
Cálcio/química , Proteínas Quinases Dependentes de AMP Cíclico/química , AMP Cíclico/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Clonagem Molecular , Cristalografia por Raios X , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Camundongos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Especificidade por Substrato , Termodinâmica
18.
J Struct Biol ; 205(2): 180-188, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30641143

RESUMO

Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR's constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca2+. FKBP12 binds to RyR1's cytoplasmic assembly 130 Šaway from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1's channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1's conformational landscape.


Assuntos
Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Ligação Proteica , Canal de Liberação de Cálcio do Receptor de Rianodina/ultraestrutura , Proteína 1A de Ligação a Tacrolimo/genética
19.
Biochem J ; 476(2): 193-209, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30530841

RESUMO

The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM-RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during titration with CaM at a wide range of Ca2+ concentrations. We showed that CaM bound to all four CaMBDs with affinities that increased with Ca2+ concentration. CaM bound to CaMBD2 and -3 with high affinities across all Ca2+ concentrations tested, but bound to CaMBD1a and -1b only at Ca2+ concentrations above 0.2 µM. Binding experiments using individual CaM domains revealed that the CaM C-domain preferentially bound to CaMBD2, and the N-domain to CaMBD3. Moreover, the Ca2+ affinity of the CaM C-domain in complex with CaMBD2 or -3 was so high that these complexes are essentially Ca2+ saturated under resting Ca2+ conditions. Conversely, the N-domain senses Ca2+ exactly in the transition from resting to activating Ca2+ when complexed to either CaMBD2 or -3. Altogether, our results support a binding model where the CaM C-domain is anchored to RyR2 CaMBD2 and saturated with Ca2+ during Ca2+ oscillations, while the CaM N-domain functions as a dynamic Ca2+ sensor that can bridge noncontiguous regions of RyR2 or clamp down onto CaMBD2.


Assuntos
Cálcio/química , Calmodulina/química , Modelos Moleculares , Miocárdio/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Calmodulina/genética , Calmodulina/metabolismo , Humanos , Domínios Proteicos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
20.
J Gen Physiol ; 151(2): 131-145, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30541771

RESUMO

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via ß-adrenergic receptor (ß-AR) activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent "hyperphosphorylation" of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation-contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch-clamp recordings and confocal Ca2+ imaging during ß-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (I Ca) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during ß-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.


Assuntos
Agonistas Adrenérgicos beta/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Isoproterenol/farmacologia , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Serina/metabolismo , Animais , Sinalização do Cálcio , Células Cultivadas , Acoplamento Excitação-Contração , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Fosforilação , Canal de Liberação de Cálcio do Receptor de Rianodina/química
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