RESUMO
Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.
Assuntos
Simportadores/química , Simportadores/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Fosforilação , Alinhamento de Sequência , Cotransportadores de K e Cl-RESUMO
Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca2+ (Ca2+o) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca2+o, deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH2-terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca2+ In 2 mM Ca2+o, ceCLHM-1 NH2-terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca2+o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Mutantes/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Canais de Cálcio/química , Canais de Cálcio/genética , Eletrofisiologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Potenciais da Membrana/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Técnicas de Patch-Clamp , Mutação Puntual , Deleção de Sequência/genéticaRESUMO
Calcium homeostasis modulator 1 (CALHM1), formerly known as FAM26C, was recently identified as a physiologically important plasma membrane ion channel. CALHM1 and its Caenorhabditis elegans homolog, CLHM-1, are regulated by membrane voltage and extracellular Ca(2+) concentration ([Ca(2+)]o). In the presence of physiological [Ca(2+)]o (â¼1.5 mM), CALHM1 and CLHM-1 are closed at resting membrane potentials but can be opened by strong depolarizations. Reducing [Ca(2+)]o increases channel open probability, enabling channel activation at negative membrane potentials. Together, voltage and Ca(2+) o allosterically regulate CALHM channel gating. Through convergent evolution, CALHM has structural features that are reminiscent of connexins and pannexins/innexins/LRRC8 (volume-regulated anion channel (VRAC)) gene families, including four transmembrane helices with cytoplasmic amino and carboxyl termini. A CALHM1 channel is a hexamer of CALHM1 monomers with a functional pore diameter of â¼14 Å. CALHM channels discriminate poorly among cations and anions, with signaling molecules including Ca(2+) and ATP able to permeate through its pore. CALHM1 is expressed in the brain where it plays an important role in cortical neuron excitability induced by low [Ca(2+)]o and in type II taste bud cells in the tongue that sense sweet, bitter, and umami tastes where it functions as an essential ATP release channel to mediate nonsynaptic neurotransmitter release. CLHM-1 is expressed in C. elegans sensory neurons and body wall muscles, and its genetic deletion causes locomotion defects. Thus, CALHM is a voltage- and Ca(2+) o-gated ion channel, permeable to large cations and anions, that plays important roles in physiology.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Conexinas/química , Conexinas/genética , Conexinas/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Transdução de SinaisRESUMO
Disruption of neuronal Ca(2+) homeostasis contributes to neurodegenerative diseases through mechanisms that are not fully understood. A polymorphism in CALHM1, a recently described ion channel that regulates intracellular Ca(2+) levels, is a possible risk factor for late-onset Alzheimer's disease. Since there are six potentially redundant CALHM family members in humans, the physiological and pathophysiological consequences of CALHM1 function in vivo remain unclear. The nematode Caenorhabditis elegans expresses a single CALHM1 homolog, CLHM-1. Here we find that CLHM-1 is expressed at the plasma membrane of sensory neurons and muscles. Like human CALHM1, C. elegans CLHM-1 is a Ca(2+)-permeable ion channel regulated by voltage and extracellular Ca(2+). Loss of clhm-1 in the body-wall muscles disrupts locomotory kinematics and biomechanics, demonstrating that CLHM-1 has a physiologically significant role in vivo. The motility defects observed in clhm-1 mutant animals can be rescued by muscle-specific expression of either C. elegans CLHM-1 or human CALHM1, suggesting that the function of these proteins is conserved in vivo. Overexpression of either C. elegans CLHM-1 or human CALHM1 in neurons is toxic, causing degeneration through a necrotic-like mechanism that is partially Ca(2+) dependent. Our data show that CLHM-1 is a functionally conserved ion channel that plays an important but potentially toxic role in excitable cell function.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Animais , Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Membrana Celular/fisiologia , Estimulação Elétrica , Humanos , Locomoção/genética , Locomoção/fisiologia , Potenciais da Membrana/fisiologia , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Oócitos/citologia , Células Receptoras Sensoriais/patologia , Células Receptoras Sensoriais/fisiologia , Especificidade da Espécie , Tato/fisiologia , Transgenes/genética , Xenopus laevisRESUMO
At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.
Assuntos
Proteínas de Caenorhabditis elegans , Receptores Nicotínicos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Levamisol/metabolismo , Levamisol/farmacologia , Paralisia , Parassimpatomiméticos , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/metabolismoRESUMO
Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium. Here, we discover the conserved ion channel CLHM-1 as a ciliary EV cargo. Imaging of EVs released from sensory neuron cilia of Caenorhabditis elegans expressing fluorescently tagged CLHM-1 and TRP polycystin-2 channel PKD-2 shows enrichment of these cargoes in distinct EV subpopulations that are differentially shed in response to mating partner availability. PKD-2 alone is present in EVs shed from the cilium distal tip, whereas CLHM-1 EVs bud from a secondary site(s), including the ciliary base. Heterotrimeric and homodimeric kinesin-2 motors have discrete impacts on PKD-2 and CLHM-1 colocalization in both cilia and EVs. Total loss of kinesin-2 activity decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde intraflagellar transport is required for selective enrichment of protein cargoes into heterogeneous EVs with different signaling potentials.
RESUMO
Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no effective treatment. Diet, as a modifiable risk factor for AD, could potentially be targeted to slow disease onset and progression. However, complexity of the human diet and indirect effects of the microbiome make it challenging to identify protective nutrients. Multiple factors contribute to AD pathogenesis, including amyloid beta (Aß) deposition, energy crisis, and oxidative stress. Here, we use Caenorhabditis elegans to define the impact of diet on Aß proteotoxicity. We discover that dietary vitamin B12 alleviates mitochondrial fragmentation, bioenergetic defects, and oxidative stress, delaying Aß-induced paralysis without affecting Aß accumulation. Vitamin B12 has this protective effect by acting as a cofactor for methionine synthase, impacting the methionine/S-adenosylmethionine (SAMe) cycle. Vitamin B12 supplementation of B12-deficient adult Aß animals is beneficial, demonstrating potential for vitamin B12 as a therapy to target pathogenic features of AD triggered by proteotoxic stress.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/farmacologia , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/efeitos dos fármacos , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Vitaminas/farmacologiaRESUMO
At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 caused levamisole hypersensitivity and had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. We also examined other genes that resulted in a levamisole-hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR-dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Levamisol/farmacologia , Músculos/metabolismo , Interferência de RNARESUMO
Chloride influx through GABA-gated chloride channels, the primary mechanism by which neural activity is inhibited in the adult mammalian brain, depends on chloride gradients established by the potassium chloride cotransporter KCC2. We used a genetic screen to identify genes important for inhibition of the hermaphrodite-specific motor neurons (HSNs) that stimulate Caenorhabditis elegans egg-laying behavior and discovered mutations in a potassium chloride cotransporter, kcc-2. Functional analysis indicates that, like mammalian KCCs, C. elegans KCC-2 transports chloride, is activated by hypotonic conditions, and is inhibited by the loop diuretic furosemide. KCC-2 appears to establish chloride gradients required for the inhibitory effects of GABA-gated and serotonin-gated chloride channels on C. elegans behavior. In the absence of KCC-2, chloride gradients appear to be altered in neurons and muscles such that normally inhibitory signals become excitatory. kcc-2 is transcriptionally upregulated in the HSN neurons during synapse development. Loss of KCC-2 produces a decrease in the synaptic vesicle population within mature HSN synapses, which apparently compensates for a lack of HSN inhibition, resulting in normal egg-laying behavior. Thus, KCC-2 coordinates the development of inhibitory neurotransmission with synapse maturation to produce mature neural circuits with appropriate activity levels.
Assuntos
Caenorhabditis elegans/fisiologia , Simportadores/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Cloretos/metabolismo , Furosemida/farmacologia , Soluções Hipotônicas , Neurônios Motores/fisiologia , Músculos/fisiologia , Mutação , Receptores Acoplados a Proteínas G/metabolismo , Homologia de Sequência , Comportamento Sexual Animal/fisiologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/genética , Vesículas Sinápticas/fisiologia , Regulação para Cima , Cotransportadores de K e Cl-RESUMO
With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a noncomplementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems.
Assuntos
Caenorhabditis elegans/genética , Técnicas de Genotipagem/métodos , Mutação Puntual , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Primers do DNA/química , Técnicas de Genotipagem/normas , Reação em Cadeia da Polimerase/normas , Sensibilidade e EspecificidadeRESUMO
To analyze mechanisms that modulate serotonin signaling, we investigated how Caenorhabditis elegans regulates the function of serotonergic motor neurons that stimulate egg-laying behavior. Egg laying is inhibited by the G protein Galphao and activated by the G protein Galphaq. We found that Galphao and Galphaq act directly in the serotonergic HSN motor neurons to control egg laying. There, the G proteins had opposing effects on transcription of the tryptophan hydroxylase gene tph-1, which encodes the rate-limiting enzyme for serotonin biosynthesis. Antiserotonin staining confirmed that Galphao and Galphaq antagonistically affect serotonin levels. Altering tph-1 gene dosage showed that small changes in tph-1 expression were sufficient to affect egg-laying behavior. Epistasis experiments showed that signaling through the G proteins has additional tph-1-independent effects. Our results indicate that (1) serotonin signaling is regulated by modulating serotonin biosynthesis and (2) Galphao and Galphaq act in the same neurons to have opposing effects on behavior, in part, by antagonistically regulating transcription of specific genes. Galphao and Galphaq have opposing effects on many behaviors in addition to egg laying and may generally act, as they do in the egg-laying system, to integrate multiple signals and consequently set levels of transcription of genes that affect neurotransmitter release.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Serotonina/biossíntese , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Caenorhabditis elegans/citologia , Regulação Enzimológica da Expressão Gênica , Neurônios Motores/citologia , Neurônios Motores/enzimologia , Neurônios Motores/metabolismo , Músculos/citologia , Músculos/enzimologia , Músculos/metabolismo , Especificidade de Órgãos , Oviposição , Regiões Promotoras Genéticas/genética , Sinapses/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismoRESUMO
Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.
Assuntos
Canais de Cálcio/fisiologia , Ativação do Canal Iônico/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores Purinérgicos/fisiologia , Percepção Gustatória/fisiologia , Paladar/fisiologia , Animais , Canais de Cálcio/análise , Feminino , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Receptores Acoplados a Proteínas G/análise , Receptores Purinérgicos/análise , Transmissão Sináptica/fisiologia , XenopusRESUMO
The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/genética , Citoplasma/metabolismo , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Técnicas de Patch-Clamp , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Like many behaviors, Caenorhabditis elegans egg laying alternates between inactive and active states. To understand how the underlying neural circuit turns the behavior on and off, we optically recorded circuit activity in behaving animals while manipulating circuit function using mutations, optogenetics, and drugs. In the active state, the circuit shows rhythmic activity phased with the body bends of locomotion. The serotonergic HSN command neurons initiate the active state, but accumulation of unlaid eggs also promotes the active state independent of the HSNs. The cholinergic VC motor neurons slow locomotion during egg-laying muscle contraction and egg release. The uv1 neuroendocrine cells mechanically sense passage of eggs through the vulva and release tyramine to inhibit egg laying, in part via the LGC-55 tyramine-gated Cl- channel on the HSNs. Our results identify discrete signals that entrain or detach the circuit from the locomotion central pattern generator to produce active and inactive states.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Canais de Cloreto/genética , Retroalimentação Fisiológica , Oviposição/genética , Receptores de Amina Biogênica/genética , Comportamento Sexual Animal/fisiologia , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cloreto/metabolismo , Colina/metabolismo , Colina/farmacologia , Feminino , Regulação da Expressão Gênica , Locomoção , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Contração Muscular/efeitos dos fármacos , Contração Muscular/genética , Optogenética , Oviposição/efeitos dos fármacos , Periodicidade , Receptores de Amina Biogênica/metabolismo , Serotonina/metabolismo , Serotonina/farmacologia , Comportamento Sexual Animal/efeitos dos fármacos , Transdução de Sinais , Tiramina/metabolismo , Tiramina/farmacologiaRESUMO
CALHM1 is a plasma membrane voltage-gated Ca2+-permeable ion channel that controls amyloid-ß (Aß) metabolism and is potentially involved in the onset of Alzheimer's disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF) â=â0.015%), but that G330D is not (MAF â=â3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca2+ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca2+ uptake and Aß levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca2+ and Aß accumulation. While these results strengthen the notion that CALHM1 influences Aß metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.
Assuntos
Doença de Alzheimer/genética , Canais de Cálcio/genética , Sinalização do Cálcio/genética , Cálcio/metabolismo , Glicoproteínas de Membrana/genética , Peptídeos beta-Amiloides , Animais , Linhagem Celular , Frequência do Gene , Células HEK293 , Humanos , XenopusRESUMO
Dysferlin is a member of the evolutionarily conserved ferlin gene family. Mutations in Dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), an inherited, progressive and incurable muscle disorder. However, the molecular mechanisms underlying disease pathogenesis are not fully understood. We found that both loss-of-function mutations and muscle-specific overexpression of C. elegans fer-1, the founding member of the Dysferlin gene family, caused defects in muscle cholinergic signaling. To determine if Dysferlin-dependent regulation of cholinergic signaling is evolutionarily conserved, we examined the in vivo physiological properties of skeletal muscle synaptic signaling in a mouse model of Dysferlin-deficiency. In addition to a loss in muscle strength, Dysferlin -/- mice also exhibited a cholinergic deficit manifested by a progressive, frequency-dependent decrement in their compound muscle action potentials following repetitive nerve stimulation, which was observed in another Dysferlin mouse model but not in a Dysferlin-independent mouse model of muscular dystrophy. Oral administration of Pyridostigmine bromide, a clinically used acetylcholinesterase inhibitor (AchE.I) known to increase synaptic efficacy, reversed the action potential defect and restored in vivo muscle strength to Dysferlin -/- mice without altering muscle pathophysiology. Our data demonstrate a previously unappreciated role for Dysferlin in the regulation of cholinergic signaling and suggest that such regulation may play a significant pathophysiological role in LGMD2B disease.