RESUMO
The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.
Assuntos
Adrenérgicos , Proteínas Monoméricas de Ligação ao GTP , Humanos , Adrenérgicos/metabolismo , Adrenérgicos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Arritmias Cardíacas/metabolismoRESUMO
L-type voltage-gated CaV1.2 channels crucially regulate cardiac muscle contraction. Activation of ß-adrenergic receptors (ß-AR) augments contraction via protein kinase A (PKA)-induced increase of calcium influx through CaV1.2 channels. To date, the full ß-AR cascade has never been heterologously reconstituted. A recent study identified Rad, a CaV1.2 inhibitory protein, as essential for PKA regulation of CaV1.2. We corroborated this finding and reconstituted the complete pathway with agonist activation of ß1-AR or ß2-AR in Xenopus oocytes. We found, and distinguished between, two distinct pathways of PKA modulation of CaV1.2: Rad dependent (â¼80% of total) and Rad independent. The reconstituted system reproduces the known features of ß-AR regulation in cardiomyocytes and reveals several aspects: the differential regulation of posttranslationally modified CaV1.2 variants and the distinct features of ß1-AR versus ß2-AR activity. This system allows for the addressing of central unresolved issues in the ß-AR-CaV1.2 cascade and will facilitate the development of therapies for catecholamine-induced cardiac pathologies.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Proteínas ras/metabolismo , Animais , Canais de Cálcio Tipo L/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Camundongos , Mutação , Miócitos Cardíacos/citologia , Oócitos/citologia , Oócitos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/genética , RNA/metabolismo , Coelhos , Receptores Adrenérgicos beta/genética , Xenopus laevis , Proteínas ras/genéticaRESUMO
Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.
Assuntos
Calmodulina , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Calmodulina/química , Família , Fosfatidilinositol 4,5-DifosfatoRESUMO
mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.
Assuntos
Chaperonina 10/química , Chaperonina 60/química , Mitocôndrias/química , Proteínas Mitocondriais/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Citosol/química , Humanos , Ligação de Hidrogênio , Hidrólise , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Dobramento de ProteínaRESUMO
Signaling cross talks between auxin, a regulator of plant development, and Ca2+, a universal second messenger, have been proposed to modulate developmental plasticity in plants. However, the underlying molecular mechanisms are largely unknown. Here, we report that in Arabidopsis roots, auxin elicits specific Ca2+ signaling patterns that spatially coincide with the expression pattern of auxin-regulated genes. We have identified the single EF-hand Ca2+-binding protein Ca2+-dependent modulator of ICR1 (CMI1) as an interactor of the Rho of plants (ROP) effector interactor of constitutively active ROP (ICR1). CMI1 expression is directly up-regulated by auxin, whereas the loss of function of CMI1 associates with the repression of auxin-induced Ca2+ increases in the lateral root cap and vasculature, indicating that CMI1 represses early auxin responses. In agreement, cmi1 mutants display an increased auxin response including shorter primary roots, longer root hairs, longer hypocotyls, and altered lateral root formation. Binding to ICR1 affects subcellular localization of CMI1 and its function. The interaction between CMI1 and ICR1 is Ca2+-dependent and involves a conserved hydrophobic pocket in CMI1 and calmodulin binding-like domain in ICR1. Remarkably, CMI1 is monomeric in solution and in vitro changes its secondary structure at cellular resting Ca2+ concentrations ranging between 10-9 and 10-8 M. Hence, CMI1 is a Ca2+-dependent transducer of auxin-regulated gene expression, which can function in a cell-specific fashion at steady-state as well as at elevated cellular Ca2+ levels to regulate auxin responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Ácidos Indolacéticos/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: Two missense mutations in KCNQ1, an imprinted gene that encodes the alpha subunit of the voltage-gated potassium channel Kv7.1, cause autosomal dominant growth hormone deficiency and maternally inherited gingival fibromatosis. We evaluated endocrine features, birth size, and subsequent somatic growth of patients with long QT syndrome 1 (LQT1) due to loss-of-function mutations in KCNQ1. DESIGN: Medical records of 104 patients with LQT1 in a single tertiary care center between 1995 and 2015 were retrospectively reviewed. METHODS: Clinical and endocrine data of the LQT1 patients were included in the analyses. RESULTS: At birth, patients with a maternally inherited mutation (n = 52) were shorter than those with paternal inheritance of the mutation (n = 29) (birth length, -0.70 ± 1.1 SDS vs. -0.2 ± 1.0 SDS, P < 0.05). Further analyses showed, however, that only newborns (n = 19) of mothers who had received beta blockers during pregnancy were shorter and lighter at birth than those with paternal inheritance of the mutation (n = 29) (-0.89 ± 1.0 SDS vs. -0.20 ± 1.0 SDS, P < 0.05; and 3,173 ± 469 vs. 3,515 ± 466 g, P < 0.05). Maternal beta blocker treatment during the pregnancy was also associated with lower cord blood TSH levels (P = 0.011) and significant catch-up growth during the first year of life (Δ0.08 SDS/month, P = 0.004). Later, childhood growth of the patients was unremarkable. CONCLUSION: Loss-of-function mutations in KCNQ1 are not associated with abnormalities in growth, whereas maternal beta blocker use during pregnancy seems to modify prenatal growth of LQT1 patients-a phenomenon followed by catch-up growth after birth.
RESUMO
Familial growth hormone deficiency provides an opportunity to identify new genetic causes of short stature. Here we combine linkage analysis with whole-genome resequencing in patients with growth hormone deficiency and maternally inherited gingival fibromatosis. We report that patients from three unrelated families harbor either of two missense mutations, c.347G>T p.(Arg116Leu) or c.1106C>T p.(Pro369Leu), in KCNQ1, a gene previously implicated in the long QT interval syndrome. Kcnq1 is expressed in hypothalamic GHRH neurons and pituitary somatotropes. Co-expressing KCNQ1 with the KCNE2 ß-subunit shows that both KCNQ1 mutants increase current levels in patch clamp analyses and are associated with reduced pituitary hormone secretion from AtT-20 cells. In conclusion, our results reveal a role for the KCNQ1 potassium channel in the regulation of human growth, and show that growth hormone deficiency associated with maternally inherited gingival fibromatosis is an allelic disorder with cardiac arrhythmia syndromes caused by KCNQ1 mutations.
Assuntos
Fibromatose Gengival/genética , Hormônio do Crescimento Humano/deficiência , Canal de Potássio KCNQ1/genética , Mutação de Sentido Incorreto , Adolescente , Hormônio Adrenocorticotrópico/metabolismo , Adulto , Alelos , Substituição de Aminoácidos , Animais , Arritmias Cardíacas/genética , Criança , Pré-Escolar , Feminino , Fibromatose Gengival/metabolismo , Humanos , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Masculino , Herança Materna/genética , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Mapas de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Adulto JovemRESUMO
In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow IKS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2) and recently we revealed the competition of PIP2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP2 and Ca2+-CaM perform the same function on IKS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca2+-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.
Assuntos
Cálcio/química , Calmodulina/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetulus , HumanosRESUMO
The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca(2+)concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca(2+) concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca(2+) CaM conformation even though four Ca(2+) ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties.
Assuntos
Calmodulina/química , Conformação Proteica , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Canais de Potássio/química , Proteínas Recombinantes/químicaRESUMO
The modulation and regulation of voltage-gated Ca(2+) channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca(2+) channel CaV1.2, α1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca(2+), presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca(2+)-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of CaV1.2 inactivation induced by Ca(2+) entry into the cell.
Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Ativação do Canal Iônico , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Membrana Celular/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Mutação/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Coelhos , Relação Estrutura-Atividade , XenopusRESUMO
The restoration of function after oncologic surgery of the oral cavity constitutes one of the major challenges facing head and neck oncology. Within the general objective of securing esthetic as well as functional reconstructions, dental rehabilitation is crucial for achieving a good outcome. Adequate dental rehabilitation allows the patient to chew food and considerably improves speech and swallowing. These reconstructions will be driven biologically or prosthetically following surgical design and outcome.
Assuntos
Neoplasias de Cabeça e Pescoço/reabilitação , Procedimentos Cirúrgicos Pré-Protéticos Bucais , Procedimentos de Cirurgia Plástica , Prostodontia/métodos , Humanos , Recuperação de Função FisiológicaRESUMO
Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.
Assuntos
Calmodulina/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/genética , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Canal de Potássio KCNQ1/metabolismo , Modelos Moleculares , Mutação , Multimerização Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo ÂnguloRESUMO
The COP9 signalosome protein complex has a central role in the regulation of development of multicellular organisms. While the function of this complex in ubiquitin-mediated protein degradation is well established, results over the past few years have hinted that the COP9 signalosome may function more broadly in the regulation of gene expression. Here, using DamID technology, we show that COP9 signalosome subunit 7 functionally associates with a large number of genomic loci in the Drosophila genome, and show that the expression of many genes within these loci is COP9 signalosome-dependent. This association is likely direct as we show CSN7 binds DNA in vitro. The genes targeted by CSN7 are preferentially enriched for transcriptionally active regions of the genome, and are involved in the regulation of distinct gene ontology groupings including imaginal disc development and cell-cycle control. In accord, loss of CSN7 function leads to cell-cycle delay and altered wing development. These results indicate that CSN7, and by extension the entire COP9 signalosome, functions directly in transcriptional control. While the COP9 signalosome protein complex has long been known to regulate protein degradation, here we expand the role of this complex by showing that subunit 7 binds DNA in vitro and functions directly in vivo in transcriptional control of developmentally important pathways that are relevant for human health.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação , Complexo do Signalossomo COP9 , Linhagem Celular , DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Loci Gênicos , Genoma de Inseto , Transcrição Gênica , Asas de Animais/crescimento & desenvolvimentoRESUMO
KCNQ1 and KCNE1 co-assembly generates the I(KS) K(+) current, which is crucial to the cardiac action potential repolarization. Mutations in their corresponding genes cause long QT syndrome (LQT) and atrial fibrillation. The A-kinase anchor protein, yotiao (also known as AKAP9), brings the I(KS) channel complex together with signaling proteins to achieve regulation upon ß1-adrenergic stimulation. Recently, we have shown that KCNQ1 helix C interacts with the KCNE1 distal C-terminus. We postulated that this interface is crucial for I(KS) channel modulation. Here, we examined the yet unknown molecular mechanisms of LQT mutations located at this intracellular intersubunit interface. All LQT mutations disrupted the internal KCNQ1-KCNE1 intersubunit interaction. LQT mutants in KCNQ1 helix C led to a decreased current density and a depolarizing shift of channel activation, mainly arising from impaired phosphatidylinositol-4,5-bisphosphate (PIP2) modulation. In the KCNE1 distal C-terminus, the LQT mutation P127T suppressed yotiao-dependent cAMP-mediated upregulation of the I(KS) current, which was caused by reduced KCNQ1 phosphorylation at S27. Thus, KCNQ1 helix C is important for channel modulation by PIP2, whereas the KCNE1 distal C-terminus appears essential for the regulation of IKS by yotiao-mediated PKA phosphorylation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canal de Potássio KCNQ1/química , Canal de Potássio KCNQ1/metabolismo , Síndrome do QT Longo/genética , Mutação de Sentido Incorreto , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Humanos , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/enzimologia , Síndrome do QT Longo/metabolismo , Fosforilação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
RGK proteins, Gem, Rad, Rem1, and Rem2, are members of the Ras superfamily of small GTP-binding proteins that interact with Ca2+ channel ß subunits to modify voltage-gated Ca2+ channel function. In addition, RGK proteins affect several cellular processes such as cytoskeletal rearrangement, neuronal dendritic complexity, and synapse formation. To probe the phylogenetic origins of RGK protein-Ca2+ channel interactions, we identified potential RGK-like protein homologs in genomes for genetically diverse organisms from both the deuterostome and protostome animal superphyla. RGK-like protein homologs cloned from Danio rerio (zebrafish) and Drosophila melanogaster (fruit flies) expressed in mammalian sympathetic neurons decreased Ca2+ current density as reported for expression of mammalian RGK proteins. Sequence alignments from evolutionarily diverse organisms spanning the protostome/deuterostome divide revealed conservation of residues within the RGK G-domain involved in RGK protein--Cavß subunit interaction. In addition, the C-terminal eleven residues were highly conserved and constituted a signature sequence unique to RGK proteins but of unknown function. Taken together, these data suggest that RGK proteins, and the ability to modify Ca2+ channel function, arose from an ancestor predating the protostomes split from deuterostomes approximately 550 million years ago.
Assuntos
Canais de Cálcio Tipo L/genética , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Proteínas ras/genética , Sequência de Aminoácidos , Animais , Canais de Cálcio Tipo L/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ratos , Ratos Wistar , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas ras/metabolismoRESUMO
Accumulation of amyloid-ß peptides (Aß), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer's disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Aß40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aß40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aß40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Multimerização Proteica , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Peptídeos beta-Amiloides/química , Animais , Cálcio/metabolismo , Células Cultivadas , Exocitose , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
DOC2B (double-C2 domain) protein is thought to be a high-affinity Ca(2+) sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca(2+)-binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure-function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca(2+) with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca(2+). In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca(2+) binding is stabilized, as reflected by the ~10-fold slower rate of Ca(2+) dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC50 of 400 nM while the C2A does not translocate at submicromolar Ca(2+) concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an ~2-fold lower EC50 and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca(2+) sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/química , Cálcio/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Domínios e Motivos de Interação entre Proteínas , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ratos , SoluçõesRESUMO
In eukaryotic Na(+)/Ca(2+) exchangers (NCX) the Ca(2+) binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca(2+) sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca(2+)-driven interdomain switch has been described, albeit how it couples Ca(2+) binding with signal propagation remains unclear. To resolve the dynamic features of Ca(2+)-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium (45)Ca(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg(2+)-bound forms of CBD12 are highly flexible, whereas Ca(2+) binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca(2+) is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca(2+)]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca(2+)-induced population shift, but the occupancy of these sites by 1 mM Mg(2+) shifts the Ca(2+) affinity (Kd) at the neighboring Ca3-Ca4 sites from â¼ 50 nM to â¼ 200 nM and thus, keeps the primary Ca(2+) sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca(2+) binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.
Assuntos
Cálcio/química , Simulação de Dinâmica Molecular , Trocador de Sódio e Cálcio/química , Animais , Sítios de Ligação , Cálcio/metabolismo , Cães , Ligação Proteica , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismoRESUMO
Voltage-dependent calcium channels (CaV) enable the inward flow of calcium currents for a wide range of cells. CaV1 and CaV2 subtype α1 subunits form the conducting pore using four repeated membrane domains connected by intracellular linkers. The domain I-II linker connects to the membrane gate (IS6), forming an α-helix, and is bound to the CaVß subunit. Previous studies indicated that this region may or may not form a continuous helix depending on the CaV subtype, thereby modulating channel activation and inactivation properties. Here, we used small-angle x-ray scattering and ensemble modeling analysis to investigate the solution structure of these linkers, extending from the membrane domain and including the CaVß-binding site, called the proximal linker (PL). The results demonstrate that the CaV1.2 PL is more flexible than the CaV2.2 PL, the flexibility is intrinsic and not dependent on CaVß binding, and the flexibility can be most easily explained by the presence of conserved glycines. Our analysis also provides a robust example of investigating protein domains in which flexibility plays an essential role.