RESUMO
The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.
Assuntos
Anoctaminas , Canais de Cloreto , Humanos , Anoctamina-1/metabolismo , Anoctamina-1/genética , Anoctaminas/metabolismo , Anoctaminas/genética , Anoctaminas/química , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Cloretos/metabolismo , Células HEK293 , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Domínios ProteicosRESUMO
Chronic obstructive pulmonary disease (COPD) manifests with a variety of clinical presentations, reflecting its complex pathology. Currently, care focuses on symptom amelioration and prevention of complications and thus is generally tailored to disease severity rather than targeting specific pathophysiologic mechanisms. Chronic inflammation and mucus hypersecretion are key features of COPD. Epithelial ion channel dysfunction may be important, as it results in airway dehydration and defective host defense, contributing to chronic airway inflammation. Recent evidence suggests considerable similarities between COPD and cystic fibrosis (CF), a disease in which chloride ion channel dysfunction has been extensively studied (in particular CFTR [CF transmembrane conductance regulator]). Understanding commonalities between CF and COPD, and the role of CFTR in CF, may help in designing strategies targeting ion channel dysfunction and lead to new treatments with potential to alter the natural history of disease progression. Here, we review the roles of airway mucus and CFTR in normal lung function, the previously underestimated contribution of mucus stasis to the development of COPD, and the evidence for targeting CFTR to counteract mucus accumulation.
Assuntos
Fibrose Cística , Doença Pulmonar Obstrutiva Crônica , Fibrose Cística/genética , Fibrose Cística/terapia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Inflamação , Transporte de Íons , Doença Pulmonar Obstrutiva Crônica/terapiaRESUMO
Ca2+-activated Cl- (ClCa) channels regulate membrane excitability and myogenic tone in vascular smooth muscles. TMEM16A-coding proteins are mainly responsible for functional ClCa channels in vascular smooth muscles, including portal vein smooth muscles (PVSMs). Caveolae are cholesterol-rich and Ω-shaped invaginations on the plasma membrane that structurally contributes to effective signal transduction. Caveolin 1 (Cav1) accumulates in caveolae to form functional complexes among receptors, ion channels, and kinases. The present study examined the functional roles of Cav1 in the expression and activity of ClCa channels in the portal vein smooth muscle cells (PVSMCs) of wild-type (WT) and Cav1-knockout (KO) mice. Contractile experiments revealed that the amplitude of spontaneous PVSM contractions was larger in Cav1-KO mice than WT mice. Under whole-cell patch-clamp configurations, ClCa currents were markedly inhibited by 1 µM Ani9 (a selective TMEM16A ClCa channel blocker) in WT and Cav1-KO PVSMCs. However, Ani9-sensitive ClCa currents were significantly larger in Cav1-KO PVSMCs than in WT PVSMCs. Expression analyses showed that TMEM16A expression levels were higher in Cav1-KO PVSMs than in WT PVSMs. Therefore, the caveolar structure formed by Cav1 negatively regulates the expression and activity of TMEM16A-mediated ClCa channels in vascular smooth muscle cells.
Assuntos
Anoctamina-1 , Caveolina 1 , Canais de Cloreto , Animais , Camundongos , Anoctamina-1/metabolismo , Cálcio/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Canais de Cloreto/genética , Camundongos Knockout , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/metabolismo , Veia Porta/metabolismoRESUMO
Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.
Assuntos
Anoctamina-1/metabolismo , Biomarcadores Tumorais/metabolismo , Sinalização do Cálcio , Carcinoma Ductal Pancreático/patologia , Fator de Crescimento Epidérmico/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Anoctamina-1/genética , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Movimento Celular , Conjuntos de Dados como Assunto , Diagnóstico Diferencial , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/mortalidade , Prognóstico , RNA Interferente Pequeno/metabolismo , RNA-Seq , Taxa de Sobrevida , Regulação para CimaRESUMO
Best Vitelliform Macular dystrophy (BVMD) is the most prevalent of the distinctive retinal dystrophies caused by mutations in the BEST1 gene. This gene, which encodes for a homopentameric calcium-activated ion channel, is crucial for the homeostasis and function of the retinal pigment epithelia (RPE), the cell type responsible for recycling the visual pigments generated by photoreceptor cells. In BVMD patients, mutations in this gene induce functional problems in the RPE cell layer with an accumulation of lipofucsin that evolves into cell death and loss of sight. In this work, we employ iPSC-RPE cells derived from a patient with the p.Pro77Ser dominant mutation to determine the correlation between this variant and the ocular phenotype. To this purpose, gene and protein expression and localization are evaluated in iPSC-RPE cells along with functional assays like phagocytosis and anion channel activity. Our cell model shows no differences in gene expression, protein expression/localization, or phagocytosis capacity, but presents an increased chloride entrance, indicating that the p.Pro77Ser variant might be a gain-of-function mutation. We hypothesize that this variant disturbs the neck region of the BEST1 channel, affecting channel function but maintaining cell homeostasis in the short term. This data shed new light on the different phenotypes of dominant mutations in BEST1, and emphasize the importance of understanding its molecular mechanisms. Furthermore, the data widen the knowledge of this pathology and open the door for a better diagnosis and prognosis of the disease.
Assuntos
Bestrofinas , Células-Tronco Pluripotentes Induzidas , Distrofia Macular Viteliforme , Bestrofinas/genética , Bestrofinas/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Epitélio Pigmentado da Retina/metabolismo , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/metabolismo , Distrofia Macular Viteliforme/patologiaRESUMO
Rationale: Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl- channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.
Assuntos
Anoctamina-1/efeitos dos fármacos , Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Depuração Mucociliar/efeitos dos fármacos , Muco/efeitos dos fármacos , Administração por Inalação , Animais , Anoctamina-1/metabolismo , Brônquios/citologia , Sinalização do Cálcio/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Técnicas de Patch-Clamp , Respiração , Mucosa Respiratória/citologia , Ovinos , Traqueia/efeitos dos fármacos , Traqueia/metabolismoRESUMO
Chloride is the major free anion in the extracellular space (>100 mM) and within the cytoplasm in eukaryotes (10 â¼ 20 mM). Cytoplasmic Cl- level is dynamically regulated by Cl- channels and transporters. It is well established that movement of Cl- across the cell membrane is coupled with cell excitability through changes in membrane potential and with water secretion. However, whether cytoplasmic Cl- plays additional roles in animal development and tissue homeostasis is unknown. Here we use genetics, cell biological and pharmacological tools to demonstrate that TMEM16A, an evolutionarily conserved calcium-activated chloride channel (CaCC), regulates cytoplasmic Cl- homeostasis and promotes plasma membrane remodeling required for mammalian epithelial morphogenesis. We demonstrate that TMEM16A-mediated control of cytoplasmic Cl- regulates the organization of the major phosphoinositide species PtdIns(4,5)P2 into microdomains on the plasma membrane, analogous to processes that cluster soluble and membrane proteins into phase-separated droplets. We further show that an adequate cytoplasmic Cl- level is required for proper endocytic trafficking and membrane supply during early stages of ciliogenesis and adherens junction remodeling. Our study thus uncovers a critical function of CaCC-mediated cytoplasmic Cl- homeostasis in controlling the organization of PtdIns(4,5)P2 microdomains and membrane remodeling. This newly defined role of cytoplasmic Cl- may shed light on the mechanisms of intracellular Cl- signaling events crucial for regulating tissue architecture and organelle biogenesis during animal development.
Assuntos
Anoctamina-1/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Morfogênese/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Junções Aderentes/genética , Junções Aderentes/metabolismo , Animais , Anoctamina-1/genética , Membrana Celular/genética , Cílios/genética , Cílios/metabolismo , Epitélio/metabolismo , Transporte de Íons/fisiologia , Camundongos , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMO
The concept that increasing airway hydration leads to improvements in mucus clearance and lung function in cystic fibrosis has been clinically validated with osmotic agents such as hypertonic saline and more convincingly with cystic fibrosis transmembrane conductance regulator (CFTR) repair therapies. Although rapidly becoming the standard of care in cystic fibrosis (CF), current CFTR modulators do not treat all patients nor do they restore the rate of decline in lung function to normal levels. As such, novel approaches are still required to ensure all with CF have effective therapies. Although CFTR plays a fundamental role in the regulation of fluid secretion across the airway mucosa, there are other ion channels and transporters that represent viable targets for future therapeutics. In this review article we will summarise the current progress with CFTR-independent approaches to restoring mucosal hydration, including epithelial sodium channel (ENaC) blockade and modulators of SLC26A9. A particular emphasis is given to modulation of the airway epithelial calcium-activated chloride channel (CaCC), TMEM16A, as there is controversy regarding whether it should be positively or negatively modulated. This is discussed in light of a recent report describing for the first time bona fide TMEM16A potentiators and their positive effects upon epithelial fluid secretion and mucus clearance.
Assuntos
Anoctamina-1/metabolismo , Fibrose Cística/metabolismo , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Animais , Ânions/metabolismo , Anoctamina-1/antagonistas & inibidores , Antiporters/metabolismo , Fibrose Cística/patologia , Descoberta de Drogas , Canais Epiteliais de Sódio/metabolismo , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Mucosa Respiratória/patologia , Transportadores de Sulfato/metabolismoRESUMO
Using the patch-clamp method in the whole cell configuration, it was shown that new conjugates of 2-aminothiophene-3-carboxylic acid with adamantane derivatives exhibit the ability to modulate CaCC activity in the single Purkinje neurons of rat cerebellum. It was noted that, depending on the nature of the substitution in the thiophene fragment, the nature of the effect on CaCC varies from inhibition to potentiation of CaCC currents. The described compounds are also blockers of the NMDA receptor ifenprodile site, which may have an additional neuroprotective contribution to the spectrum of biological activity of these compounds.
Assuntos
Adamantano/farmacologia , Canais de Cloreto/antagonistas & inibidores , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Células Cultivadas , Masculino , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
This study explored the mechanism by which Ca2+-activated Cl- channels (CaCCs) encoded by the Tmem16a gene are regulated by calmodulin-dependent protein kinase II (CaMKII) and protein phosphatases 1 (PP1) and 2A (PP2A). Ca2+-activated Cl- currents (IClCa) were recorded from HEK-293 cells expressing mouse TMEM16A. IClCa were evoked using a pipette solution in which free Ca2+ concentration was clamped to 500 nM, in the presence (5 mM) or absence of ATP. With 5 mM ATP, IClCa decayed to <50% of the initial current magnitude within 10 min after seal rupture. IClCa rundown seen with ATP-containing pipette solution was greatly diminished by omitting ATP. IClCa recorded after 20 min of cell dialysis with 0 ATP were more than twofold larger than those recorded with 5 mM ATP. Intracellular application of autocamtide-2-related inhibitory peptide (5 µM) or KN-93 (10 µM), two specific CaMKII inhibitors, produced a similar attenuation of TMEM16A rundown. In contrast, internal application of okadaic acid (30 nM) or cantharidin (100 nM), two nonselective PP1 and PP2A blockers, promoted the rundown of TMEM16A in cells dialyzed with 0 ATP. Mutating serine 528 of TMEM16A to an alanine led to a similar inhibition of TMEM16A rundown to that exerted by either one of the two CaMKII inhibitors tested, which was not observed for three putative CaMKII consensus sites for phosphorylation (T273, T622, and S730). Our results suggest that TMEM16A-mediated CaCCs are regulated by CaMKII and PP1/PP2A. Our data also suggest that serine 528 of TMEM16A is an important contributor to the regulation of IClCa by CaMKII.
Assuntos
Anoctamina-1/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Anoctamina-1/metabolismo , Benzilaminas/farmacologia , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cantaridina/farmacologia , Cloretos/metabolismo , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Proteínas de Neoplasias/metabolismo , Ácido Okadáico/farmacologia , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Sulfonamidas/farmacologiaRESUMO
Current therapies including pharmaceutical intervention and surgery have limited efficacy on stress urinary incontinence (SUI). One type of SUI is due to low intraurethral pressure caused by the disabled contraction of urethral smooth muscle (USM). However, the molecular mechanisms underlying the motility of USM remain unknown. Here, we show that USM represents spontaneous tone after stretching in humans and mice. Deletion of TMEM16A in the smooth muscle of mice abolishes spontaneous urethral tone. Furthermore, ClCa currents and [Ca2+ ]i in TMEM16ASMKO mice were largely impaired. Inhibitors of ryanodine receptor (RyR), TMEM16A encoded calcium-activated chloride channel (ClCa ) and L-type voltage-dependent calcium channel (VDCC) fully prevented spontaneous tone accompanied by a significant decrease of intracellular calcium concentration ([Ca2+ ]i ). In summary, RyR-ClCa -VDCC signaling contributes to spontaneous USM tone. This finding may provide a new promising approach for women with stress SUI who reject surgery.
Assuntos
Anoctamina-1/metabolismo , Canais de Cálcio/metabolismo , Tono Muscular/fisiologia , Músculo Liso/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Uretra/metabolismo , Animais , Canais de Cloreto/metabolismo , Feminino , Humanos , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Incontinência Urinária por Estresse/metabolismoRESUMO
Lateral inhibitory feedback from horizontal cells (HCs) to cones establishes center-surround receptive fields and color opponency in the retina. When HCs hyperpolarize to light, inhibitory feedback to cones increases activation of cone Ca2+ currents (ICa) that can in turn activate additional currents. We recorded simultaneously from cones and HCs to analyze cone currents activated by HC feedback in salamander retina. Depolarization-activated inward tail currents in cones were inhibited by CaCCinh-A01 that inhibits both Ano1 and Ano2 Ca2+-activated Cl- currents (ICl(Ca)). An Ano1-selective inhibitor Ani9 was less effective suggesting that Ano2 is the predominant ICl(Ca) subtype in cones. CaCCinh-A01 inhibited feedback currents more strongly when intracellular Ca2+ in cones was buffered with 0.05â¯mM EGTA compared to stronger buffering with 5â¯mM EGTA. By contrast, blocking glutamate transporter anion currents (ICl(Glu)) with TBOA had stronger inhibitory effects on cone feedback currents when Ca2+ buffering was strong. Inward feedback currents ran down at rates intermediate between rundown of glutamate release and ICl(Ca), consistent with contributions to feedback from both ICl(Ca) and ICl(Glu). These results suggest that Cl- channels coupled to glutamate transporters help to speed inward feedback currents initiated by local changes in intracellular [Ca2+] close to synaptic ribbons of cones whereas Ano2 Ca2+-activated Cl- channels contribute to slower components of feedback regulated by spatially extensive changes in intracellular [Ca2+].
Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Cálcio/metabolismo , Cloretos/metabolismo , Potenciais da Membrana/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinapses/metabolismo , Ambystoma , Animais , Modelos Animais , Técnicas de Patch-Clamp , Células Fotorreceptoras Retinianas Cones/citologiaRESUMO
Seminal vesicles (SVs), a pair of male accessory glands, contract upon sympathetic nerve excitation during ejaculation while developing spontaneous phasic constrictions in the inter-ejaculatory storage phase. Recently, the fundamental role of the mucosa in generating spontaneous activity in SV of the guinea pig has been revealed. Stretching the mucosa-intact but not mucosa-denuded SV smooth muscle evokes spontaneous phasic contractions arising from action potential firing triggered by electrical slow waves and associated Ca2+ flashes. These spontaneous events primarily depend on sarco-endoplasmic reticulum (SR/ER) Ca2+ handling linked with the opening of Ca2+-activated chloride channels (CaCCs) resulting in the generation of slow waves. Slow waves in mucosa-intact SV smooth muscle are abolished upon blockade of gap junctions, suggesting that seminal smooth muscle cells are driven by cells distributed in the mucosa. In the SV mucosal preparations dissected free from the smooth muscle layer, a population of cells located just beneath the epithelium develop spontaneous Ca2+ transients relying on SR/ER Ca2+ handling. In the lamina propria of the SV mucosa, vimentin-immunoreactive interstitial cells including platelet-derived growth factor receptor α (PDGFRα)-immunoreactive cells are distributed, while known pacemaker cells in other smooth muscle tissues, e.g. c-Kit-positive interstitial cells or α-smooth muscle actin-positive atypical smooth muscle cells, are absent. The spontaneously-active subepithelial cells appear to drive spontaneous activity in SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. Interstitial cells in the lamina propria may act as intermediaries of signal transmission from the subepithelial cells to the smooth muscle cells.
Assuntos
Células Intersticiais de Cajal/fisiologia , Mucosa/fisiologia , Contração Muscular , Músculo Liso/fisiologia , Glândulas Seminais/fisiologia , Animais , Sinalização do Cálcio , Cobaias , Canais Iônicos/fisiologia , Masculino , Miócitos de Músculo Liso/fisiologiaRESUMO
Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A "Ca2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.
Assuntos
Bestrofinas/química , Bestrofinas/metabolismo , Cálcio/metabolismo , Galinhas/metabolismo , Alanina/genética , Substituição de Aminoácidos , Animais , Bestrofinas/genética , Canais de Cloreto/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Olfactory transduction in vertebrate olfactory receptor neurons (ORNs) involves primarily a cAMP-signaling cascade that leads to the opening of cyclic-nucleotide-gated (CNG), nonselective cation channels. The consequent Ca2+ influx triggers adaptation but also signal amplification, the latter by opening a Ca2+-activated Cl channel (ANO2) to elicit, unusually, an inward Cl current. Hence the olfactory response has inward CNG and Cl components that are in rapid succession and not easily separable. We report here success in quantitatively separating these two currents with respect to amplitude and time course over a broad range of odorant strengths. Importantly, we found that the Cl current is the predominant component throughout the olfactory dose-response relation, down to the threshold of signaling to the brain. This observation is very surprising given a recent report by others that the olfactory-signal amplification effected by the Ca2+-activated Cl current does not influence the behavioral olfactory threshold in mice.
Assuntos
Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Olfato/fisiologia , Animais , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Camundongos , Odorantes/análise , Neurônios Receptores Olfatórios/fisiologia , Transdução de Sinais , Olfato/genéticaRESUMO
Calcium-activated chloride channels (CaCCs) are key players in transepithelial ion transport and fluid secretion, smooth muscle constriction, neuronal excitability, and cell proliferation. The CaCC regulator 1 (CLCA1) modulates the activity of the CaCC TMEM16A/Anoctamin 1 (ANO1) by directly engaging the channel at the cell surface, but the exact mechanism is unknown. Here we demonstrate that the von Willebrand factor type A (VWA) domain within the cleaved CLCA1 N-terminal fragment is necessary and sufficient for this interaction. TMEM16A protein levels on the cell surface were increased in HEK293T cells transfected with CLCA1 constructs containing the VWA domain, and TMEM16A-like currents were activated. Similar currents were evoked in cells exposed to secreted VWA domain alone, and these currents were significantly knocked down by TMEM16A siRNA. VWA-dependent TMEM16A modulation was not modified by the S357N mutation, a VWA domain polymorphism associated with more severe meconium ileus in cystic fibrosis patients. VWA-activated currents were significantly reduced in the absence of extracellular Mg2+, and mutation of residues within the conserved metal ion-dependent adhesion site motif impaired the ability of VWA to potentiate TMEM16A activity, suggesting that CLCA1-TMEM16A interactions are Mg2+- and metal ion-dependent adhesion site-dependent. Increase in TMEM16A activity occurred within minutes of exposure to CLCA1 or after a short treatment with nocodazole, consistent with the hypothesis that CLCA1 stabilizes TMEM16A at the cell surface by preventing its internalization. Our study hints at the therapeutic potential of the selective activation of TMEM16A by the CLCA1 VWA domain in loss-of-function chloride channelopathies such as cystic fibrosis.
Assuntos
Canais de Cloreto/metabolismo , Magnésio/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Substituição de Aminoácidos , Anoctamina-1 , Linhagem Celular , Canais de Cloreto/genética , Humanos , Proteínas de Neoplasias/genética , Domínios Proteicos , Estabilidade ProteicaRESUMO
Calcium-activated chloride channels (CaCCs) are a family of anionic transmembrane ion channels. They are mainly responsible for the movement of Cl- and other anions across the biological membranes, and they are widely expressed in different tissues. Since the Cl- flow into or out of the cell plays a crucial role in hyperpolarizing or depolarizing the cells, respectively, the impact of intracellular Ca2+ concentration on these channels is attracting a lot of attentions. After summarizing the molecular, biophysical, and pharmacological properties of CaCCs, the role of CaCCs in normal cellular functions will be discussed, and I will emphasize how dysregulation of CaCCs in pathological conditions can account for different diseases. A better understanding of CaCCs and a pivotal regulatory role of Ca2+ can shed more light on the therapeutic strategies for different neurological disorders that arise from chloride dysregulation, such as asthma, cystic fibrosis, and neuropathic pain.
Assuntos
Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Asma/tratamento farmacológico , Asma/metabolismo , Cálcio/metabolismo , Canais de Cloreto/química , Canais de Cloreto/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl- is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl- gradient and Cl- channels were investigated by substitution of extracellular Cl- with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl- channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. About 100-300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl- movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl- being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl- channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.
Assuntos
Anoctamina-1/metabolismo , Cloretos/metabolismo , Vasos Linfáticos/metabolismo , Contração Muscular , Miócitos de Músculo Liso/metabolismo , Idoso , Animais , Anoctamina-1/antagonistas & inibidores , Feminino , Humanos , Vasos Linfáticos/fisiologia , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa/metabolismoRESUMO
BACKGROUND: In a previous study utilizing mass spectrometry-based proteomics, we identified calcium-activated chloride channel regulator 1 (CLCA1) as a potential tumor suppressor in pancreatic cancer and the expression was inversely correlated with patient survival. The aim of the study was to further validate the prognostic significance of CLCA1 in pancreatic cancer. METHODS: CLCA1 expression was evaluated with tissue microarrays and immunohistochemistry in 140 patients with pancreatic ductal adenocarcinoma that underwent surgical resection at Skåne University Hospital, Sweden. Kaplan-Meier and Cox proportional hazards modeling were used to explore the association between CLCA1 and clinicopathological factors and survival. RESULTS: CLCA1 expression was denoted as positive in 90 tumors (64.3%), with positive staining being limited to the tumor cells. There were no significant association between CLCA1 expression and established clinicopathological parameters. Low CLCA1 expression correlated significantly with shorter disease-free survival (11.9 vs 17.5 months, P = 0.042). Multivariable Cox regression analysis confirmed the results (HR 0.61, 95% CI-0.40-0.92, P = 0.019). CONCLUSIONS: Low CLCA1 expression is an independent factor of poor disease-free survival in pancreatic cancer.
Assuntos
Biomarcadores Tumorais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Canais de Cloreto/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/patologia , Canais de Cloreto/metabolismo , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Pancreáticas/patologia , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
Ca(2+)-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca(2+) is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach--channel inactivation induced by membrane-tethering of an associated protein (ChIMP)--we discovered that Ca(2+)-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca(2+)-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM1234. These effects are Ca(2+)-dependent sensitization of activation (CDSA) and Ca(2+)-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca(2+)-activated chloride channels.