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1.
Proc Natl Acad Sci U S A ; 117(44): 27502-27508, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33087577

RESUMO

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNß expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.


Assuntos
Adenosina/metabolismo , Células Epiteliais/metabolismo , Imunidade Inata , Imunidade nas Mucosas , Nucleotídeos Cíclicos/metabolismo , 5'-Nucleotidase/metabolismo , Linhagem Celular Tumoral , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/imunologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Interferon beta/metabolismo , Mucosa Intestinal/citologia , Diester Fosfórico Hidrolases/metabolismo , Poli I-C/imunologia , Pirofosfatases/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais/imunologia
2.
Am J Physiol Renal Physiol ; 319(6): F1027-F1036, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33103446

RESUMO

Similar to other organs, renal lymphatics remove excess fluid, solutes, and macromolecules from the renal interstitium. Given the kidney's unique role in maintaining body fluid homeostasis, renal lymphatics may be critical in this process. However, little is known regarding the pathways involved in renal lymphatic vessel function, and there are no studies on the effects of drugs targeting impaired interstitial clearance, such as diuretics. Using pressure myography, we showed that renal lymphatic collecting vessels are sensitive to changes in transmural pressure and have an optimal range of effective pumping. In addition, they are responsive to vasoactive factors known to regulate tone in other lymphatic vessels including prostaglandin E2 and nitric oxide, and their spontaneous contractility requires Ca2+ and Cl-. We also demonstrated that Na+-K+-2Cl- cotransporter Nkcc1, but not Nkcc2, is expressed in extrarenal lymphatic vessels. Furosemide, a loop diuretic that inhibits Na+-K+-2Cl- cotransporters, induced a dose-dependent dilation in lymphatic vessels and decreased the magnitude and frequency of spontaneous contractions, thereby reducing the ability of these vessels to propel lymph. Ethacrynic acid, another loop diuretic, had no effect on vessel tone. These data represent a significant step forward in our understanding of the mechanisms underlying renal lymphatic vessel function and highlight potential off-target effects of furosemide that may exacerbate fluid accumulation in edema-forming conditions.


Assuntos
Rim/anatomia & histologia , Rim/fisiologia , Vasos Linfáticos/fisiologia , Animais , Cálcio/metabolismo , Cloretos/metabolismo , Furosemida/farmacologia , Vasos Linfáticos/efeitos dos fármacos , Masculino , NG-Nitroarginina Metil Éster , Óxido Nítrico/metabolismo , Pressão , Ratos , Ratos Sprague-Dawley , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia
3.
PLoS Genet ; 16(10): e1009100, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33085659

RESUMO

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Fibrose Cística/terapia , Processamento de RNA/genética , Processamento Alternativo/genética , Substituição de Aminoácidos/genética , Cloretos/metabolismo , Fibrose Cística/patologia , Eletromiografia , Éxons/genética , Variação Genética/genética , Células HEK293 , Humanos , Íntrons/genética , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Nucleotídeos/genética , Medicina de Precisão/métodos , Cultura Primária de Células , RNA Mensageiro/genética
4.
Nat Commun ; 11(1): 3935, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769979

RESUMO

GABAA/glycine-mediated neuronal inhibition critically depends on intracellular chloride (Cl-) concentration which is mainly regulated by the K+-Cl- co-transporter 2 (KCC2) in the adult central nervous system (CNS). KCC2 heterogeneity thus affects information processing across CNS areas. Here, we uncover a gradient in Cl- extrusion capacity across the superficial dorsal horn (SDH) of the spinal cord (laminae I-II: LI-LII), which remains concealed under low Cl- load. Under high Cl- load or heightened synaptic drive, lower Cl- extrusion is unveiled in LI, as expected from the gradient in KCC2 expression found across the SDH. Blocking TrkB receptors increases KCC2 in LI, pointing to differential constitutive TrkB activation across laminae. Higher Cl- lability in LI results in rapidly collapsing inhibition, and a form of activity-dependent synaptic plasticity expressed as a continuous facilitation of excitatory responses. The higher metaplasticity in LI as compared to LII differentially affects sensitization to thermal and mechanical input. Thus, inconspicuous heterogeneity of Cl- extrusion across laminae critically shapes plasticity for selective nociceptive modalities.


Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Cloretos/metabolismo , Plasticidade Neuronal/fisiologia , Nociceptividade/fisiologia , Células do Corno Posterior/fisiologia , Animais , Células Cultivadas , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Neurológicos , Optogenética , Cultura Primária de Células , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptor trkB/antagonistas & inibidores , Receptor trkB/metabolismo , Simportadores/metabolismo
5.
Nat Commun ; 11(1): 4320, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859916

RESUMO

In autosomal dominant polycystic kidney disease (ADPKD) multiple bilateral renal cysts gradually enlarge, leading to a decline in renal function. Transepithelial chloride secretion through cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1) are known to drive cyst enlargement. Here we demonstrate that loss of Pkd1 increased expression of TMEM16A and CFTR and Cl- secretion in murine kidneys, with TMEM16A essentially contributing to cyst growth. Upregulated TMEM16A enhanced intracellular Ca2+ signaling and proliferation of Pkd1-deficient renal epithelial cells. In contrast, increase in Ca2+ signaling, cell proliferation and CFTR expression was not observed in Pkd1/Tmem16a double knockout mice. Knockout of Tmem16a or inhibition of TMEM16A in vivo by the FDA-approved drugs niclosamide and benzbromarone, as well as the TMEM16A-specific inhibitor Ani9 largely reduced cyst enlargement and abnormal cyst cell proliferation. The present data establish a therapeutic concept for the treatment of ADPKD.


Assuntos
Anoctamina-1/genética , Anoctamina-1/metabolismo , Cistos/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismo , Animais , Anoctamina-1/efeitos dos fármacos , Benzobromarona/farmacologia , Canais de Cálcio , Proliferação de Células , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística , Cistos/tratamento farmacológico , Cistos/genética , Modelos Animais de Doenças , Cães , Células Epiteliais/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Néfrons/metabolismo , Niclosamida/farmacologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética
6.
Phytochemistry ; 179: 112464, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32798746

RESUMO

A phytochemical study on the root extracts of Neorautanenia mitis, a Nigerian medicinal plant used in the management of diarrhea, led to the isolation of one new and 19 known natural products. These compounds and crude extracts were evaluated for Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel and calcium-activated Cl- channel (TMEM16A) inhibitory activities in T84 and Calu-3 cells, respectively. Four compounds namely dolineon, neodulin, pachyrrhizine, and neotenone inhibited cAMP-induced Cl- secretion across T84 cell monolayers with IC50 values of ~0.81 µM, ~2.42 µM, ~2.87 µM, and ~4.66 µM, respectively. Dolineon having the highest inhibitory activity also inhibited a Ca + activated Cl- channel (TMEM16A) with an IC50 value of ~4.38 µM. The in vitro antidiarrheal activity of dolineon was evaluated on cholera toxin (CT) induced chloride secretion in T84 cells, where it inhibited CT-induced chloride secretion by >70% at 100 µM. Dolineon also inhibited CT-induced fluid secretion by ~70% in an in vivo mouse closed loop model at a dose of 16.9 µg/loop. The cytotoxicity of the extracts and compounds was evaluated on KB, Vero and BHK21 cells, dolineon showed low cytotoxicity of >29.6 µM and 57.30 + 6.77 µM against Vero and BHK21 cells, respectively. Our study revealed that several compounds isolated from N. mitis showed antidiarrheal activity. The most active compound dolineon can potentially serve as a lead compound towards the development of CFTR and TMEM16A inhibitors as future therapeutics for secretory diarrhea.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Chumbo , Animais , Transporte Biológico , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia/tratamento farmacológico , Camundongos
7.
Plant Physiol Biochem ; 155: 161-168, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32758997

RESUMO

Maize has to avoid excess tissue accumulation of Cl- to withstand conditions of Cl--salinity. Restriction of loading of Cl- into the root xylem is one mechanism to keep shoot Cl--concentrations low. The proportion of Cl- that reaches the shoot has to be stored away from the primary site of photosynthesis and growth. We tested whether or not maize is able to re-translocate significant amounts of Cl- from shoot back to root and out into the rooting media. Ion analysis revealed that maize cannot re-translocate Cl-; however, it is stored in sheaths of the old leaves and, surprisingly, in roots. Sequestration of Cl- in the roots might be a strategy to keep concentrations low in young growing shoot tissues and in leaf blades where photosynthesis is running.


Assuntos
Cloretos/metabolismo , Raízes de Plantas/fisiologia , Salinidade , Zea mays/fisiologia , Brotos de Planta/fisiologia , Xilema/fisiologia
8.
Am J Physiol Cell Physiol ; 319(4): C641-C656, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726161

RESUMO

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl- between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN-), a property that does not correspond with any of the Cl- channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN- permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl- channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN- conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN--selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.


Assuntos
Canais de Cloreto/genética , Antiportadores de Cloreto-Bicarbonato/genética , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/genética , Transportadores de Sulfato/genética , Animais , Ânions/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Pigmentos da Retina/metabolismo , Tiocianatos/metabolismo
9.
Clin Nucl Med ; 45(10): 805-807, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32604118

RESUMO

A 56-year-old man with metastatic castration-resistant prostate cancer was referred for radioligand therapy with Lu-prostate-specific membrane antigen. In the third cycle, a posttherapy whole-body scan showed unexpected skeletal and joint uptake apart from his known metastatic lesions. This observation raised suspicion for possible impurity (mainly free lutetium) in the applied radiopharmaceutical product. After contacting the radiopharmaceutical company, we were informed that the radiochemical purity of the used batch of Lu-prostate-specific membrane antigen had been 95%. This is the first report of excess free lutetium scan pattern and its complications in a patient undergoing radioligand therapy.


Assuntos
Cloretos/metabolismo , Dipeptídeos , Compostos Heterocíclicos com 1 Anel , Neoplasias de Próstata Resistentes à Castração/diagnóstico por imagem , Neoplasias de Próstata Resistentes à Castração/patologia , Imagem Corporal Total , Artefatos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias de Próstata Resistentes à Castração/metabolismo , Resultado do Tratamento
10.
Am J Physiol Renal Physiol ; 319(3): F534-F540, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32715757

RESUMO

Cl--sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT). Cl- enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl- channel ClC-K2 or K+-Cl- cotransporter (KCC). While KCC is electroneutral, Cl- exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+ channel activity is expected to influence Cl- movement across the basolateral membrane. Although a role for intracellular Cl- in the regulation of WNK and NCC has been established, intracellular Cl- concentrations ([Cl-]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl-]i in DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl- fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl-]i was ~7 mM. Stimulation of Cl- exit with low-Cl- hypotonic solutions decreased [Cl-]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl-]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl-]i . Blockade of basolateral K+ channels (Kir4.1/5.1) with barium significantly increased [Cl-]i. Finally, a decrease in extracellular K+ concentration transiently decreased [Cl-]i, whereas raising extracellular K+ transiently increased [Cl-]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl-]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl-]i in the DCT.


Assuntos
Cloretos/metabolismo , Túbulos Renais Distais/fisiologia , Canais de Potássio/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Cloretos/química , Fenômenos Eletrofisiológicos , Camundongos , Imagem Molecular , Simportadores de Cloreto de Sódio/genética
11.
Nucleic Acids Res ; 48(13): 7454-7467, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520327

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Processamento de RNA , Aminofenóis/farmacologia , Brônquios/citologia , Linhagem Celular Tumoral , Células Cultivadas , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Mutação , Quinolonas/farmacologia
12.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G142-G150, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567323

RESUMO

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.


Assuntos
Canais de Cloreto/metabolismo , Colo/fisiologia , Mucosa Intestinal/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Canais de Cloreto/genética , Cloretos/metabolismo , Feminino , Transporte de Íons , Masculino , Técnicas de Patch-Clamp , Canais de Potássio/genética , Transporte Proteico , Ratos , Ratos Sprague-Dawley
14.
Am J Physiol Cell Physiol ; 319(2): C371-C380, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32579473

RESUMO

Cation-coupled chloride cotransporters (CCC) play a role in modulating intracellular chloride concentration ([Cl-]i) and cell volume. Cell shrinkage and cell swelling are accompanied by an increase or decrease in [Cl-]i, respectively. Cell shrinkage and a decrease in [Cl-]i increase the activity of NKCCs (Na-K-Cl cotransporters: NKCC1, NKCC2, and Na-Cl) and inhibit the activity of KCCs (K-Cl cotransporters: KCC1 to KCC4), wheras cell swelling and an increase in [Cl-]i activate KCCs and inhibit NKCCs; thus, it is unlikely that the same kinase is responsible for both effects. WNK1 and WNK4 are chloride-sensitive kinases that modulate the activity of CCC in response to changes in [Cl-]i. Here, we showed that WNK3, another member of the serine-threonine kinase WNK family with known effects on CCC, is not sensitive to [Cl-]i but can be regulated by changes in extracellular tonicity. In contrast, WNK4 is highly sensitive to [Cl-]i but is not regulated by changes in cell volume. The activity of WNK3 toward NaCl cotransporter is not affected by eliminating the chloride-binding site of WNK3, further confirming that the kinase is not sensitive to chloride. Chimeric WNK3/WNK4 proteins were produced, and analysis of the chimeras suggests that sequences within the WNK's carboxy-terminal end may modulate the chloride affinity. We propose that WNK3 is a cell volume-sensitive kinase that translates changes in cell volume into phosphorylation of CCC.


Assuntos
Tamanho Celular , Proteínas Serina-Treonina Quinases/genética , Simportadores de Cloreto de Sódio/metabolismo , Proteínas de Xenopus/genética , Animais , Cloretos/química , Cloretos/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Humanos , Oócitos/química , Oócitos/metabolismo , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/química , Xenopus/genética , Xenopus/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
15.
Science ; 368(6493): 893-897, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32439795

RESUMO

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.


Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Ácido Glutâmico/metabolismo , Vesículas Sinápticas/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/química , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Concentração de Íons de Hidrogênio , Transporte de Íons , Potenciais da Membrana , Domínios Proteicos , Ratos
16.
Sci Rep ; 10(1): 8395, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32439841

RESUMO

We highlight mechanical stretching and bending of membranes and the importance of membrane deformations in the analysis of swelling dynamics of biological systems, including cells and subcellular organelles. Membrane deformation upon swelling generates tensile stress and internal pressure, contributing to volume changes in biological systems. Therefore, in addition to physical (internal/external) and chemical factors, mechanical properties of the membranes should be considered in modeling analysis of cellular swelling. Here we describe an approach that considers mechanical properties of the membranes in the analysis of swelling dynamics of biological systems. This approach includes membrane bending and stretching deformations into the model, producing a more realistic description of swelling. We also discuss the effects of membrane stretching on swelling dynamics. We report that additional pressure generated by membrane bending is negligible, compared to pressures generated by membrane stretching, when both membrane surface area and volume are variable parameters. Note that bending deformations are reversible, while stretching deformation may be irreversible, leading to membrane disruption when they exceed a certain threshold level. Therefore, bending deformations need only be considered in reversible physiological swelling, whereas stretching deformations should also be considered in pathological irreversible swelling. Thus, the currently proposed approach may be used to develop a detailed biophysical model describing the transition from physiological to pathological swelling mode.


Assuntos
Membrana Celular/química , Modelos Biológicos , Fenômenos Biomecânicos , Membrana Celular/fisiologia , Cloretos/química , Cloretos/metabolismo , Simulação por Computador , Membrana Eritrocítica/química , Membrana Eritrocítica/fisiologia , Sódio/química , Sódio/metabolismo
17.
Am J Physiol Regul Integr Comp Physiol ; 319(1): R96-R105, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32459971

RESUMO

The rectal gland of the spiny dogfish Squalus acanthias secretes a salt solution isosmotic with plasma that maintains the salt homeostasis of the fish. It secretes salt against an electrochemical gradient that requires the expenditure of energy. Isolated rectal glands perfused without glucose secrete salt, albeit at a rate about 30% of glands perfused with 5 mM glucose. Gradually reducing the glucose concentration is associated with a progressive decrease in the secretion of chloride. The apparent Km for the exogenous glucose-dependent chloride secretion is around 2 mM. Phloretin and cytochalasin B, agents that inhibit facilitated glucose carriers of the solute carrier 2 (Slc2) family such as glucose transporter 2 (GLUT2), do not inhibit the secretion of chloride by the perfused rectal glands. Phloridzin, which inhibits Slc5 family of glucose symporters, or α-methyl-d-glucoside, which competitively inhibits the uptake of glucose through Slc5 symporters, inhibit the secretion of chloride. Thus the movement of glucose into the rectal gland cells appears to be mediated by a sodium-glucose symporter. Sodium-glucose cotransporter 1 (SGLT1), the first member of the Slc5 family of sodium-linked glucose symporters, was cloned from the rectal gland. No evidence of GLUT2 was found. The persistence of secretion of chloride in the absence of glucose in the perfusate suggests that there is an additional source of energy within the cells. The use of 2-mercapto-acetate did not result in any change in the secretion of chloride, suggesting that the oxidation of fatty acids is not the source of energy for the secretion of chloride. Perfusion of isolated glands with KCN in the absence of glucose further reduces the secretion of chloride but does not abolish it, again suggesting that there is another source of energy within the cells. Glucose was measured in the rectal gland cells and found to be at concentrations in the range of that in the perfusate. Glycogen measurements indicated that there are significant stores of glucose in the rectal gland. Moreover, glycogen synthase was partially cloned from rectal gland cells. The open reading frame of glycogen phosphorylase was also cloned from rectal gland cells. Measurements of glycogen phosphorylase showed that the enzyme is mostly in its active form in the cells. The cells of the rectal gland of the spiny dogfish require exogenous glucose to fully support the active secretion of salt. They have the means to transport glucose into the cells in the form of SGLT1. The cells also have an endogenous supply of glucose as glycogen and have the necessary elements to synthesize, store, and hydrolyze it.


Assuntos
Cloretos/metabolismo , Glucose/metabolismo , Glândula de Sal/metabolismo , Squalus/metabolismo , Animais , Sequência de Bases , Glucose/farmacologia , Transportador de Glucose Tipo 2/metabolismo , Glicogênio/metabolismo , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/metabolismo , Homeostase , Técnicas In Vitro , Cianeto de Potássio/farmacologia , Glândula de Sal/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II/metabolismo
18.
PLoS Comput Biol ; 16(3): e1007530, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226009

RESUMO

This work reports a dynamical Markov state model of CLC-2 "fast" (pore) gating, based on 600 microseconds of molecular dynamics (MD) simulation. In the starting conformation of our CLC-2 model, both outer and inner channel gates are closed. The first conformational change in our dataset involves rotation of the inner-gate backbone along residues S168-G169-I170. This change is strikingly similar to that observed in the cryo-EM structure of the bovine CLC-K channel, though the volume of the intracellular (inner) region of the ion conduction pathway is further expanded in our model. From this state (inner gate open and outer gate closed), two additional states are observed, each involving a unique rotameric flip of the outer-gate residue GLUex. Both additional states involve conformational changes that orient GLUex away from the extracellular (outer) region of the ion conduction pathway. In the first additional state, the rotameric flip of GLUex results in an open, or near-open, channel pore. The equilibrium population of this state is low (∼1%), consistent with the low open probability of CLC-2 observed experimentally in the absence of a membrane potential stimulus (0 mV). In the second additional state, GLUex rotates to occlude the channel pore. This state, which has a low equilibrium population (∼1%), is only accessible when GLUex is protonated. Together, these pathways model the opening of both an inner and outer gate within the CLC-2 selectivity filter, as a function of GLUex protonation. Collectively, our findings are consistent with published experimental analyses of CLC-2 gating and provide a high-resolution structural model to guide future investigations.


Assuntos
Canais de Cloreto/genética , Ativação do Canal Iônico/fisiologia , Animais , Bovinos , Cloretos/metabolismo , Biologia Computacional/métodos , Cinética , Cadeias de Markov , Potenciais da Membrana , Modelos Biológicos , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação
19.
PLoS Genet ; 16(4): e1008643, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294086

RESUMO

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl- cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.


Assuntos
Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/genética , Mutação , Domínios Proteicos/genética , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloretos/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/congênito , Surdez/genética , Éxons/genética , Feminino , Expressão Gênica , Células HEK293 , Humanos , Lactente , Macaca fascicularis , Masculino , Linhagem , Processamento de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
20.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244302

RESUMO

The treatment of cystic fibrosis (CF) patients homozygous for the F508del mutation with Orkambi®, a combination of a corrector (lumacaftor) and a potentiator (ivacaftor) of the mutated CFTR protein, resulted in some amelioration of the respiratory function. However, a great variability in the clinical response was also observed. The aim of this study was to evaluate the response to Orkambi® in a small cohort of F508del/F508del patients (n = 14) in terms of clinical and laboratory parameters, including ex vivo CFTR activity in mononuclear cells (MNCs), during a 12-month treatment. Patients responded with an increase in percent predicted forced expiratory volume in 1 s (FEV1%) and body mass index (BMI) as well as with a decrease in white blood cell (WBC) total counts and serum C-reactive protein (CRP) levels, although not significantly. Sweat chloride and CFTR-dependent chloride efflux were found to decrease and increase, respectively, as compared with pre-therapy values. CFTR and BMI showed a statistically significant correlation during Orkambi® treatment. Clustering analysis showed that CFTR, BMI, sweat chloride, FEV1%, and WBC were strongly associated. These data support the notion that CFTR-dependent chloride efflux in MNCs should be investigated as a sensitive outcome measure of Orkambi® treatment in CF patients.


Assuntos
Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Fibrose Cística/genética , Fibrose Cística/terapia , Leucócitos/metabolismo , Quinolonas/uso terapêutico , Adolescente , Adulto , Índice de Massa Corporal , Criança , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Combinação de Medicamentos , Feminino , Volume Expiratório Forçado , Homozigoto , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Mutação , Pacientes , Testes de Função Respiratória , Adulto Jovem
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