RESUMEN
Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.
Asunto(s)
Mutación con Ganancia de Función , Infertilidad Masculina , Receptores de Inositol 1,4,5-Trifosfato , Animales , Calcio/metabolismo , Células HEK293 , Humanos , Infertilidad Masculina/genética , Inositol 1,4,5-Trifosfato , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Ratones , Mutación/genéticaRESUMEN
The monocot lineage-specific miR528 was previously established as a multistress regulator. However, it remains largely unclear how miR528 participates in response to salinity stress in rice. Here, we show that miR528 positively regulates rice salt tolerance by down-regulating a gene encoding l-ascorbate oxidase (AO), thereby bolstering up the AO-mediated abscisic acid (ABA) synthesis and ROS scavenging. Overexpression of miR528 caused a substantial increase in ascorbic acid (AsA) and ABA contents but a significant reduction in ROS accumulation, resulting in the enhanced salt tolerance of rice plants. Conversely, knockdown of miR528 or overexpression of AO stimulated the expression of the AO gene, hence lowering the level of AsA, a critical antioxidant that promotes the ABA content but reduces the ROS level, and then compromising rice tolerance to salinity. Together, the findings reveal a novel mechanism of the miR528-AO module-mediated salt tolerance by modulating the processes of AsA and ABA metabolism as well as ROS detoxification, which adds a new regulatory role to the miR528-AO stress defense pathway in rice.
Asunto(s)
Ácido Abscísico/metabolismo , Ácido Ascórbico/metabolismo , MicroARNs/genética , Oryza , Tolerancia a la Sal , Ascorbato Oxidasa , Regulación de la Expresión Génica de las Plantas , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tolerancia a la Sal/genética , Estrés FisiológicoRESUMEN
K+-dependent Na+-Ca2+ exchangers (NCKXs) play an important role in Ca2+ homeostasis in many tissues. NCKX proteins are bi-directional plasma membrane Ca2+-transporters which utilize the inward Na+ and outward K+ gradients to move Ca2+ ions into and out of the cytosol (4Na+:1Ca2+â¯+â¯1â¯K+). In this study, we carried out scanning mutagenesis of all the residues of the highly conserved α-1 and α-2 repeats of NCKX2 to identify residues important for K+ transport. These structural elements are thought to be critical for cation transport. Using fluorescent intracellular Ca2+-indicating dyes, we measured the K+ dependence of transport carried out by wildtype or mutant NCKX2 proteins expressed in HEK293 cells and analyzed shifts in the apparent binding affinity (Km) of mutant proteins in comparison with the wildtype exchanger. Of the 93 residue substitutions tested, 34 were found to show a significant shift in the external K+ ion dependence of which 16 showed an increased affinity to K+ ions and 18 showed a decreased affinity and hence are believed to be important for K+ ion binding and transport. We also identified 8 residue substitutions that resulted in a partial loss of K+ dependence. Our biochemical data provide strong support for the cation binding sites identified in a homology model of NCKX2 based on crystal structures reported for distantly related archaeal Na+-Ca2+ exchanger NCX_Mj. In addition, we compare our results here with our previous studies that report on residues important for Ca2+ and Na+ binding. Supported by CIHR MOP-81327.
Asunto(s)
Potasio/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Células HEK293 , Humanos , Transporte Iónico/fisiologíaRESUMEN
PKA (protein kinase A)-dependent phosphorylation of the cardiac Ca2+-release channel/RyR2 (type 2 ryanodine receptor)is believed to directly dissociate FKBP12.6 (12.6 kDa FK506-binding protein) from the channel, causing abnormal channel activation and Ca2+ release. To gain insight into the structural basis of the regulation of RyR2 by PKA, we determined the three-dimensional location of the PKA site Ser2030. GFP (green fluorescent protein) was inserted into RyR2-wt (wild-type RyR2)and RyR2 mutant, A4860G, after Thr2023. The resultant GFP-RyR2 fusion proteins, RyR2T2023-GFP and RyR2(A4860G)T2023-GFP, were expressed in HEK-293 (human embryonic kidney) cells and functionally characterized. Ca2+-release assays revealed that both GFP-RyR2 fusion proteins formed caffeine- and ryanodine-sensitive Ca2+-release channels. Further analyses using[3H]ryanodine binding demonstrated that the insertion of GFPinto RyR2-wt after Thr2023 reduced the sensitivity of the channelto activation by Ca2+ or caffeine. RyR2(A4860G)T2023-GFP was found to be structurally more stable than RyR2T2023-GFP and was subsequently used as a basis for three-dimensional reconstruction. Cryo-electronmicroscopy and single particle image processing of the purified RyR2(A4860G)T2023-GFP protein revealed the location of the inserted GFP, and hence the Ser2030 PKA site in domain 4,a region that may be involved in signal transduction between the transmembrane and cytoplasmic domains. Like the Ser2808 PKA site reported previously, the Ser2030 site is not located close to the FKBP12.6-binding site mapped previously, indicating that neither of these PKA sites is directly involved in FKBP12.6 binding. On the basis of the three-dimensional localizations of a number of residues or regions, a model for the subunit organization in the structure of RyR2 is proposed.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Corazón/fisiología , Canal Liberador de Calcio Receptor de Rianodina/química , Serina , Cafeína/farmacología , Calcio/metabolismo , Calcio/farmacología , Línea Celular , Microscopía por Crioelectrón , Humanos , Procesamiento de Imagen Asistido por Computador , Riñón/embriología , Modelos Moleculares , Miocardio/enzimología , Fosforilación , Polimorfismo de Nucleótido Simple , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , TransfecciónRESUMEN
The phosphorylation of the cardiac Ca(2+)-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.
Asunto(s)
Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Línea Celular , Ventrículos Cardíacos/patología , Humanos , Isoproterenol/farmacología , Modelos Biológicos , Mutación , Miocitos Cardíacos/metabolismo , Fosforilación , Ratas , Serina/químicaRESUMEN
We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma(-/-)) mice have enhanced cardiac contractility attributable to cAMP-dependent increases in sarcoplasmic reticulum (SR) Ca(2+) content and release but not L-type Ca(2+) current (I(Ca,L)), demonstrating PI3Kgamma locally regulates cAMP levels in cardiomyocytes. Because phosphodiesterases (PDEs) can contribute to cAMP compartmentation, we examined whether the PDE activity was altered by PI3Kgamma ablation. Selective inhibition of PDE3 or PDE4 in wild-type (WT) cardiomyocytes elevated Ca(2+) transients, SR Ca(2+) content, and phospholamban phosphorylation (PLN-PO(4)) by similar amounts to levels observed in untreated PI3Kgamma(-/-) myocytes. Combined PDE3 and PDE4 inhibition caused no further increases in SR function. By contrast, only PDE3 inhibition affected Ca(2+) transients, SR Ca(2+) loads, and PLN-PO(4) levels in PI3Kgamma(-/-) myocytes. On the other hand, inhibition of PDE3 or PDE4 alone did not affect I(Ca,L) in either PI3Kgamma(-/-) or WT cardiomyocytes, whereas simultaneous PDE3 and PDE4 inhibition elevated I(Ca,L) in both groups. Ryanodine receptor (RyR(2)) phosphorylation levels were not different in basal conditions between PI3Kgamma(-/-) and WT myocytes and increased in both groups with PDE inhibition. Our results establish that L-type Ca(2+) channels, RyR(2), and SR Ca(2+) pumps are regulated differently in distinct subcellular compartments by PDE3 and PDE4. In addition, the loss of PI3Kgamma selectively abolishes PDE4 activity, not PDE3, in subcellular compartments containing the SR Ca(2+)-ATPase but not RyR(2) or L-type Ca(2+) channels.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Miocitos Cardíacos/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/metabolismo , Compartimento Celular/fisiología , Fosfatidilinositol 3-Quinasa Clase Ib , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Inhibidores Enzimáticos/farmacología , Cardiopatías/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Mutantes , Contracción Miocárdica/fisiología , Miocitos Cardíacos/citología , Fosfatidilinositol 3-Quinasas/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
OBJECTIVE: To prepare beta-cyclodextrin polymer (beta-CDP) beads using as a drug carrier and study the release behaviors of Berberine Hydrochlorrde (BH) in beta-CDP beads. METHODS: beta-CDP beads were synthesized by inverse emulsion polymerization. The inclusion complex between beta-CD and BH were investigated by phase solubility method. And the release of BH in vitro was studied by UV spectra. RESULTS: Inclusion compounds of beta-CD and BH with 1: 1 molar ratio were found under the condition of pH 1.4 and pH 7.4, with corresponding stability constants of 48.85 L/mol and 122.11 L/mol, respectively. Finally, the controlled release behavior of BH in polymer beads were studied and the release rate of BH kept almost constant after 24 h. Release rate of BH was higher at pH 1.4 than that at pH 7.4. CONCLUSION: beta-CDP beads may be an ideal controlled drug release carrier.
Asunto(s)
Celulosa/química , Ciclodextrinas/química , Portadores de Fármacos/química , Celulosa/síntesis química , Ciclodextrinas/síntesis química , Preparaciones de Acción Retardada , Portadores de Fármacos/síntesis química , Liberación de Fármacos , Concentración de Iones de Hidrógeno , SolubilidadRESUMEN
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.
Asunto(s)
Modelos Moleculares , Proteínas Musculares/química , Miocardio/química , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/química , Serina/química , Proteínas de Unión a Tacrolimus/química , Animales , Anticuerpos Monoclonales/química , Arritmias Cardíacas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Microscopía por Crioelectrón , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Epítopos/química , Humanos , Ratones , Proteínas Musculares/metabolismo , Miocardio/metabolismo , Mapeo Peptídico , Fosforilación , Unión Proteica/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Serina/metabolismo , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH(2)-terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH(2)-terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. The resultant GFP-RyR2 fusion protein, RyR2(S437-GFP,) was expressed in HEK293 cells and characterized using Ca(2+) release, [(3)H]ryanodine binding, and single cell Ca(2+) imaging studies. These functional analyses revealed that RyR2(S437-GFP) forms a caffeine- and ryanodine-sensitive Ca(2+) release channel that possesses Ca(2+) and caffeine dependence of activation indistinguishable from that of wild type (wt) RyR2. HEK293 cells expressing RyR2(S437-GFP) displayed a propensity for store overload-induced Ca(2+) release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2(S437-GFP) was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2(S437-GFP) revealed the location of the inserted GFP, and hence the NH(2)-terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH(2)-terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.