RESUMO
Our brains consist of 80% water, which is continuously shifted between different compartments and cell types during physiological and pathophysiological processes. Disturbances in brain water homeostasis occur with pathologies such as brain oedema and hydrocephalus, in which fluid accumulation leads to elevated intracranial pressure. Targeted pharmacological treatments do not exist for these conditions owing to our incomplete understanding of the molecular mechanisms governing brain water transport. Historically, the transmembrane movement of brain water was assumed to occur as passive movement of water along the osmotic gradient, greatly accelerated by water channels termed aquaporins. Although aquaporins govern the majority of fluid handling in the kidney, they do not suffice to explain the overall brain water movement: either they are not present in the membranes across which water flows or they appear not to be required for the observed flow of water. Notably, brain fluid can be secreted against an osmotic gradient, suggesting that conventional osmotic water flow may not describe all transmembrane fluid transport in the brain. The cotransport of water is an unconventional molecular mechanism that is introduced in this Review as a missing link to bridge the gap in our understanding of cellular and barrier brain water transport.
Assuntos
Encéfalo/metabolismo , Água/metabolismo , Animais , Aquaporinas/metabolismo , Água Corporal/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Tamanho Celular , Líquido Cefalorraquidiano/metabolismo , Endotélio Vascular/metabolismo , Líquido Extracelular/metabolismo , Sistema Glinfático/fisiologia , Humanos , Líquido Intracelular/metabolismo , Transporte de Íons , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Osmose , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Espaço SubaracnóideoRESUMO
Volume regulation is essential for cell homeostasis and physiological function. Amongst the sensory molecules that have been associated with volume regulation is the transient receptor potential vanilloid 4 (TRPV4), which is a non-selective cation channel that in conjunction with aquaporins, typically controls regulatory volume decrease (RVD). Here we show that the interaction between orthologous AQP4 (Aqp4a) and TRPV4 (Trpv4) is important for regulatory volume increase (RVI) in post-activated marine fish spermatozoa under high osmotic stress. Based upon electrophysiological, volumetric, and in vivo and ex vivo functional experiments using the pharmacological and immunological inhibition of Aqp4a and Trpv4 our model suggests that upon ejaculation and exposure to the hypertonic seawater, spermatozoon shrinkage is initially mediated by water efflux through Aqp1aa in the flagellar tail. The shrinkage results in an increase in intracellular Ca2+ concentration, and the activation of sperm motility and a Na+/K+/2Cl- (NKCC1) cotransporter. The activity of NKCC1 is required for the initiation of cell swelling, which secondarily activates the Aqp4a-Trpv4 complex to facilitate the influx of water via Aqp4a-M43 and Ca2+ via Trpv4 and L-type channels for the mediation of RVI. The inhibitory experiments show that blocking of each of these events prevents either shrinkage or RVI. Our data thus reveal that post-activated marine fish spermatozoa are capable of initiating RVI under a high hypertonic stress, which is essential for the maintenance of sperm motility.
Assuntos
Tamanho Celular , Pressão Osmótica , Motilidade dos Espermatozoides , Espermatozoides , Canais de Cátion TRPV , Animais , Masculino , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Espermatozoides/metabolismo , Motilidade dos Espermatozoides/fisiologia , Aquaporina 4/metabolismo , Aquaporina 4/genética , Cálcio/metabolismo , Peixes/metabolismo , Peixes/fisiologia , Natação , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genéticaRESUMO
BACKGROUND: The molecular mechanisms underlying development of posthemorrhagic hydrocephalus (PHH) following subarachnoid hemorrhage (SAH) remain incompletely understood. Consequently, treatment strategies tailored towards the individual patient remain limited. This study aimed to identify proteomic cerebrospinal fluid (CSF) biomarkers capable of predicting shunt dependency and functional outcome in patients with SAH in order to improve informed clinical decision making. METHODS: Ventricular CSF samples were collected twice from 23 patients with SAH who required external ventricular drain (EVD) insertion (12 patients with successful EVD weaning, 11 patients in need of permanent CSF shunting due to development of PHH). The paired CSF samples were collected acutely after ictus and later upon EVD removal. Cisternal CSF samples were collected from 10 healthy control subjects undergoing vascular clipping of an unruptured aneurysm. All CSF samples were subjected to mass spectrometry-based proteomics analysis. Proteomic biomarkers were quantified using area under the curve (AUC) estimates from a receiver operating curve (ROC). RESULTS: CSF from patients with SAH displayed a distinct proteomic profile in comparison to that of healthy control subjects. The CSF collected acutely after ictus from patients with SAH was moreover distinct from that collected weeks later but appeared similar in the weaned and shunted patient groups. Sixteen unique proteins were identified as potential predictors of shunt dependency, while three proteins were identified as potential predictors of functional outcome assessed six months after ictus with the modified Rankin Scale. CONCLUSIONS: We here identified several potential proteomic biomarkers in CSF from patients with SAH capable of predicting (i) shunt dependency and thus development of PHH and (ii) the functional outcome assessed six months after ictus. These proteomic biomarkers may have the potential to aid clinical decision making by predicting shunt dependency and functional outcome following SAH.
RESUMO
Cerebral edema is a life-threatening condition that can cause permanent brain damage or death if left untreated. Existing therapies aim at mitigating the associated elevated intracranial pressure, yet they primarily alleviate pressure rather than prevent edema formation. Prophylactic anti-edema therapy necessitates novel drugs targeting edema formation. Aquaporin 4 (AQP4), an abundantly expressed water pore in mammalian glia and ependymal cells, has been proposed to be involved in cerebral edema formation. A series of novel compounds have been tested for their potential inhibitory effects on AQP4. However, selectivity, toxicity, functional inhibition, sustained therapeutic concentration, and delivery into the central nervous system are major challenges. Employing extensive density-functional theory (DFT) calculations, we identified a previously unreported thermodynamically stable tautomer of the recently identified AQP4-specific inhibitor TGN-020 (2-(nicotinamide)-1,3,4-thiadiazol). This novel form, featuring a distinct hydrogen-bonding pattern, served as a template for a COSMOsim-3D-based virtual screen of proprietary compounds from Origenis™. The screening identified ORI-TRN-002, an electronic homologue of TGN-020, demonstrating high solubility and low protein binding. Evaluating ORI-TRN-002 on AQP4-expressing Xenopus laevis oocytes using a high-resolution volume recording system revealed an IC50 of 2.9 ± 0.6 µM, establishing it as a novel AQP4 inhibitor. ORI-TRN-002 exhibits superior solubility and overcomes free fraction limitations compared to other reported AQP4 inhibitors, suggesting its potential as a promising anti-edema therapy for treating cerebral edema in the future.
Assuntos
Aquaporina 4 , Edema Encefálico , Niacinamida , Tiadiazóis , Animais , Aquaporina 4/antagonistas & inibidores , Edema , Niacinamida/análogos & derivadosRESUMO
BACKGROUND: The molecular mechanisms underlying development of posthemorrhagic hydrocephalus (PHH) remain elusive. The aim of this systematic review was to evaluate existing literature on increased CSF secretion and impaired CSF absorption as pathogenic contributors to CSF accumulation in neonatal and adult PHH. METHODS: The systematic review was conducted in accordance with the PRISMA guidelines. Relevant studies published before March 11th, 2023, were identified from PubMed and reference lists. Studies were screened for eligibility using predefined inclusion and exclusion criteria. Data from eligible studies were extracted and potential sources of bias were evaluated. RESULTS: Nineteen studies quantified CSF production rates and/or CSF absorption capacity in human patients with PHH or animals with experimentally induced PHH. Increased CSF production was reported as early as 24 h and as late as 28 days post ictus in six out of eight studies quantifying CSF production rates in animals with experimentally induced PHH. Impaired CSF absorption was reported in all four studies quantifying CSF absorption capacity in human patients with PHH and in seven out of nine studies quantifying CSF absorption capacity in animals with experimentally induced PHH. Impaired CSF absorption was reported as early as 30 min and as late as 10 months post ictus. CONCLUSIONS: The pathological CSF accumulation in PHH likely arises from a combination of increased CSF secretion and impaired CSF absorption, which may manifest at different time scales following a hemorrhagic event. Emergent evidence on increased CSF secretion by the choroid plexus may herald a paradigm shift in our understanding of PHH.
Assuntos
Hemorragia Cerebral , Hidrocefalia , Recém-Nascido , Animais , Humanos , Hemorragia Cerebral/complicações , Hidrocefalia/etiologia , Plexo CorióideoRESUMO
BACKGROUND: By applying an unbiased proteomic approach, we aimed to search for cerebrospinal fluid (CSF) protein biomarkers distinguishing between obstructive and communicating hydrocephalus in order to improve appropriate surgical selection for endoscopic third ventriculostomy vs. shunt implants. Our second study purpose was to look for potential CSF biomarkers distinguishing between patients with adult chronic hydrocephalus benefitting from surgery (responders) vs. those who did not (non-responders). METHODS: Ventricular CSF samples were collected from 62 patients with communicating hydrocephalus and 28 patients with obstructive hydrocephalus. CSF was collected in relation to the patients' surgical treatment. As a control group, CSF was collected from ten patients with unruptured aneurysm undergoing preventive surgery (vascular clipping). RESULTS: Mass spectrometry-based proteomic analysis of the samples identified 1251 unique proteins. No proteins differed significantly between the communicating hydrocephalus group and the obstructive hydrocephalus group. Four proteins were found to be significantly less abundant in CSF from communicating hydrocephalus patients compared to control subjects. A PCA plot revealed similar proteomic CSF profiles of obstructive and communicating hydrocephalus and control samples. For obstructive hydrocephalus, ten proteins were found to predict responders from non-responders. CONCLUSION: Here, we show that the proteomic profile of ventricular CSF from patients with hydrocephalus differs slightly from control subjects. Furthermore, we find ten predictors of response to surgical outcome (endoscopic third ventriculostomy or ventriculo-peritoneal shunt) in patients with obstructive hydrocephalus.
Assuntos
Hidrocefalia , Terceiro Ventrículo , Adulto , Humanos , Proteômica , Hidrocefalia/cirurgia , Ventriculostomia/efeitos adversos , Resultado do Tratamento , Biomarcadores , Terceiro Ventrículo/cirurgiaRESUMO
The molecular mechanisms underlying the development of posthemorrhagic hydrocephalus (PHH) remain incompletely understood. As the disease pathogenesis often cannot be attributed to visible cerebrospinal fluid (CSF) drainage obstructions, we here aimed to elucidate whether elevated CSF osmolality following subarachnoid hemorrhage (SAH) could potentiate the formation of ventricular fluid, and thereby contribute to the pathological CSF accumulation observed in PHH. The CSF osmolality was determined in 32 patients with acute SAH after external ventricular drainage (EVD) placement and again upon EVD removal and compared with the CSF osmolality from 14 healthy control subjects undergoing vascular clipping of an unruptured aneurism. However, we found no evidence of elevated CSF osmolality or electrolyte concentration in patients with SAH when compared to that of healthy control subjects. We detected no difference in CSF osmolality and electrolyte content in patients with successful EVD weaning versus those that were shunted due to PHH. Taken together, elevated CSF osmolality does not appear to underlie the development of PHH following SAH. The pathological CSF accumulation observed in this patient group must thus instead be attributed to other pathological alterations associated with the abnormal presence of blood within the CSF compartments following SAH.
Assuntos
Hidrocefalia , Hemorragia Subaracnóidea , Humanos , Derivações do Líquido Cefalorraquidiano/efeitos adversos , Hidrocefalia/etiologia , Procedimentos Neurocirúrgicos/efeitos adversos , Hemorragia Subaracnóidea/complicaçõesRESUMO
Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers. Depleting free membrane cholesterol with m-ß-cyclodextrin (MßCD) augmented TRPV4 activation by the agonist GSK1016790A, swelling and strain, with the effects reversed by cholesterol supplementation. MßCD increased membrane expression of TRPV4, caveolin-1, and flotillin. TRPV4 did not colocalize or interact with caveolae or lipid rafts, apart from a truncated â¼75 kDa variant partially precipitated by a caveolin-1 antibody. MßCD induced currents in TRPV4-expressing Xenopus laevis oocytes. Thus, membrane cholesterol regulates trabecular transduction of mechanical information, with TRPV4 channels mainly located outside the cholesterol-enriched membrane domains. Moreover, the biomechanical milieu itself shapes the lipid content of TM membranes. Diet, cholesterol metabolism, and mechanical stress might modulate the conventional outflow pathway and intraocular pressure in glaucoma and diabetes in part by modulating TM mechanosensing.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Citoesqueleto/metabolismo , Canais de Cátion TRPV/metabolismo , Idoso , Animais , Membrana Celular/química , Células Cultivadas , Humanos , Masculino , Mecanotransdução Celular , Canais de Cátion TRPV/genética , Xenopus laevisRESUMO
The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4-deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4-/- mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4-expressing Xenopus laevis oocytes monitored by a high-resolution volume recording system, we here demonstrate that the compound TGN-020 is such a selective AQP4 inhibitor. TGN-020 targets the tested species of AQP4 with an IC50 of ~3.5 µM, but displays no inhibitory effect on the other AQPs (AQP1-AQP9). With this tool, we employed rat hippocampal slices and ion-sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN-020-mediated inhibition of AQP4 did not prevent stimulus-induced extracellular space shrinkage, nor did it slow clearance of the activity-evoked K+ transient. These data, obtained with a verified isoform-selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K+ clearance or the associated extracellular space shrinkage.
Assuntos
Neuroglia , Animais , Aquaporina 4/genética , Aquaporinas , Astrócitos/metabolismo , Edema , Camundongos , Neuroglia/metabolismo , Isoformas de Proteínas , Ratos , Água/metabolismoAssuntos
Aquaporina 4 , Encéfalo , Aquaporina 4/metabolismo , Encéfalo/metabolismo , Homeostase , HumanosRESUMO
KEY POINTS: The large-pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C-terminal cleavage led to a Panx1 open-state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1-mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. ABSTRACT: Pannexin 1 (Panx1) is a large-pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant-selectivity of Panx1 activation and inhibition, we employed Panx1-expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C-terminal truncation or cell shrinkage elevated Panx1-mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+ ]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl- , lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1-mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant-selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.
Assuntos
Conexinas/fisiologia , Canais Iônicos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Corantes Fluorescentes , Ácido Glutâmico , Células HEK293 , Humanos , Ácido Láctico , Oócitos , Xenopus laevisRESUMO
Many retinal diseases are associated with pathological cell swelling, but the underlying etiology remains to be established. A key component of the volume-sensitive machinery, the transient receptor potential vanilloid 4 (TRPV4) ion channel, may represent a sensor and transducer of cell swelling, but the molecular link between the swelling and TRPV4 activation is unresolved. Here, our results from experiments using electrophysiology, cell volumetric measurements, and fluorescence imaging conducted in murine retinal cells and Xenopus oocytes indicated that cell swelling in the physiological range activated TRPV4 in Müller glia and Xenopus oocytes, but required phospholipase A2 (PLA2) activity exclusively in Müller cells. Volume-dependent TRPV4 gating was independent of cytoskeletal rearrangements and phosphorylation. Our findings also revealed that TRPV4-mediated transduction of volume changes is dependent by its N terminus, more specifically by its distal-most part. We conclude that the volume sensitivity and function of TRPV4 in situ depend critically on its functional and cell type-specific interactions.
Assuntos
Células Ependimogliais/metabolismo , Ativação do Canal Iônico/fisiologia , Neuroglia/metabolismo , Oócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Tamanho Celular , Células Ependimogliais/citologia , Feminino , Ativação do Canal Iônico/genética , Camundongos , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Técnicas de Patch-Clamp , Fosfolipases A2/metabolismo , Fosforilação , Ratos , Canais de Cátion TRPV/genética , Xenopus laevisRESUMO
Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.
Assuntos
Conexina 43/química , Conexina 43/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Fenômenos Eletrofisiológicos , Simulação de Dinâmica Molecular , Permeabilidade , Porosidade , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Especificidade por SubstratoRESUMO
Neuronal signaling in the central nervous system (CNS) associates with release of K+ into the extracellular space resulting in transient increases in [K+ ]o . This elevated K+ is swiftly removed, in part, via uptake by neighboring glia cells. This process occurs in parallel to the [K+ ]o elevation and glia cells thus act as K+ sinks during the neuronal activity, while releasing it at the termination of the pulse. The molecular transport mechanisms governing this glial K+ absorption remain a point of debate. Passive distribution of K+ via Kir4.1-mediated spatial buffering of K+ has become a favorite within the glial field, although evidence for a quantitatively significant contribution from this ion channel to K+ clearance from the extracellular space is sparse. The Na+ /K+ -ATPase, but not the Na+ /K+ /Cl- cotransporter, NKCC1, shapes the activity-evoked K+ transient. The different isoform combinations of the Na+ /K+ -ATPase expressed in glia cells and neurons display different kinetic characteristics and are thereby distinctly geared toward their temporal and quantitative contribution to K+ clearance. The glia cell swelling occurring with the K+ transient was long assumed to be directly associated with K+ uptake and/or AQP4, although accumulating evidence suggests that they are not. Rather, activation of bicarbonate- and lactate transporters appear to lead to glial cell swelling via the activity-evoked alkaline transient, K+ -mediated glial depolarization, and metabolic demand. This review covers evidence, or lack thereof, accumulated over the last half century on the molecular mechanisms supporting activity-evoked K+ and extracellular space dynamics.
Assuntos
Espaço Extracelular , Espaço Extracelular/metabolismo , Neuroglia/metabolismo , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
KEY POINTS: Neuronal activity induces fluctuation in extracellular space volume, [K+ ]o and pHo , the management of which influences neuronal function The neighbour astrocytes buffer the K+ and pH and swell during the process, causing shrinkage of the extracellular space In the present study, we report the developmental rise of the homeostatic control of the extracellular space dynamics, for which regulation becomes tighter with maturation and thus is proposed to ensure efficient synaptic transmission in the mature animals The extracellular space dynamics of volume, [K+ ]o and pHo evolve independently with developmental maturation and, although all of them are inextricably tied to neuronal activity, they do not couple directly. ABSTRACT: Neuronal activity in the mammalian central nervous system associates with transient extracellular space (ECS) dynamics involving elevated K+ and pH and shrinkage of the ECS. These ECS properties affect membrane potentials, neurotransmitter concentrations and protein function and are thus anticipated to be under tight regulatory control. It remains unresolved to what extent these ECS dynamics are developmentally regulated as synaptic precision arises and whether they are directly or indirectly coupled. To resolve the development of homeostatic control of [K+ ]o , pH, and ECS and their interaction, we utilized ion-sensitive microelectrodes in electrically stimulated rat hippocampal slices from rats of different developmental stages (postnatal days 3-28). With the employed stimulation paradigm, the stimulus-evoked peak [K+ ]o and pHo transients were stable across age groups, until normalized to neuronal activity (field potential amplitude), in which case the K+ and pH shifted significantly more in the younger animals. By contrast, ECS dynamics increased with age until normalized to the field potential, and thus correlated with neuronal activity. With age, the animals not only managed the peak [K+ ]o better, but also displayed swifter post-stimulus removal of [K+ ]o , in correlation with the increased expression of the α1-3 isoforms of the Na+ /K+ -ATPase, and a swifter return of ECS volume. The different ECS dynamics approached a near-identical temporal pattern in the more mature animals. In conclusion, although these phenomena are inextricably tied to neuronal activity, our data suggest that they do not couple directly.
Assuntos
Espaço Extracelular/fisiologia , Hipocampo/fisiologia , Potássio/fisiologia , Envelhecimento/fisiologia , Animais , Estimulação Elétrica , Feminino , Concentração de Íons de Hidrogênio , Masculino , Neurônios/fisiologia , Ratos Sprague-DawleyRESUMO
Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca2+-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (Asp-50, Asp-172, and Asp-179) have been implicated previously in the Ca2+ sensitivity of other hemichannel isoforms. Although the aspartate at position Asp-50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner and underscores that hemichannels are selective rather than non-discriminating and freely diffusable pores.
Assuntos
Conexina 30/metabolismo , Junções Comunicantes/fisiologia , Ativação do Canal Iônico , Substituição de Aminoácidos , Animais , Canais de Cálcio , Conexina 30/genética , Etídio/metabolismo , Humanos , Íons/metabolismo , Permeabilidade , Xenopus laevis/genéticaRESUMO
KEY POINTS: Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume-sensing ion channel irrespective of the origin of the cell swelling. ABSTRACT: Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non-selective cation channel permeable to Ca2+ , it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+ -dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4-AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume-sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co-expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4-mediated transmembrane currents. Similar TRPV4 activation was induced by co-expression of a cognate channel, AQP1. The level of osmotically-induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co-expressed water-translocating Na+ /K+ /2Cl- cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume-dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume-sensor, rather than as an osmo-sensor.
Assuntos
Tamanho Celular , Pressão Osmótica , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Potenciais de Ação , Animais , Aquaporinas/metabolismo , Ratos , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , XenopusRESUMO
The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3ß and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation.
Assuntos
Proteínas 14-3-3/metabolismo , Aquaporina 2/metabolismo , Regulação da Expressão Gênica , Animais , Biotinilação , Desamino Arginina Vasopressina/química , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Rim/metabolismo , Túbulos Renais/metabolismo , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ressonância de Plasmônio de Superfície , Ubiquitina/metabolismo , Vasopressinas/metabolismoRESUMO
Ammonia is a biologically potent molecule, and the regulation of ammonia levels in the mammalian body is, therefore, strictly controlled. The molecular paths of ammonia permeation across plasma membranes remain ill-defined, but the structural similarity of water and NH3 has pointed to the aquaporins as putative NH3-permeable pores. Accordingly, a range of aquaporins from mammals, plants, fungi, and protozoans demonstrates ammonia permeability. Aquaporin 4 (AQP4) is highly expressed at perivascular glia end-feet in the mammalian brain and may, with this prominent localization at the blood-brain-interface, participate in the exchange of ammonia, which is required to sustain the glutamate-glutamine cycle. Here we observe that AQP4-expressing Xenopus oocytes display a reflection coefficient <1 for NH4Cl at pH 8.0, at which pH an increased amount of the ammonia occurs in the form of NH3 Taken together with an NH4Cl-mediated intracellular alkalization (or lesser acidification) of AQP4-expressing oocytes, these data suggest that NH3 is able to permeate the pore of AQP4. Exposure to NH4Cl increased the membrane currents to a similar extent in uninjected oocytes and in oocytes expressing AQP4, indicating that the ionic NH4 (+) did not permeate AQP4. Molecular dynamics simulations revealed partial pore permeation events of NH3 but not of NH4 (+) and a reduced energy barrier for NH3 permeation through AQP4 compared with that of a cholesterol-containing lipid bilayer, suggesting AQP4 as a favored transmembrane route for NH3 Our data propose that AQP4 belongs to the growing list of NH3-permeable water channels.
Assuntos
Amônia/química , Amônia/metabolismo , Aquaporina 4/química , Aquaporina 4/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Cloreto de Amônio/química , Cloreto de Amônio/metabolismo , Animais , Aquaporina 4/genética , Aquaporinas/química , Aquaporinas/genética , Aquaporinas/metabolismo , Canais Iônicos/genética , Transporte de Íons/fisiologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Simulação de Dinâmica Molecular , Oócitos , Ratos , Xenopus laevisRESUMO
During neuronal activity in the mammalian brain, the K+ released into the synaptic space is initially buffered by the astrocytic compartment. In parallel, the extracellular space (ECS) shrinks, presumably due to astrocytic cell swelling. With the Na+ /K+ /2Cl- cotransporter and the Kir4.1/AQP4 complex not required for the astrocytic cell swelling in the hippocampus, the molecular mechanisms underlying the activity-dependent ECS shrinkage have remained unresolved. To identify these molecular mechanisms, we employed ion-sensitive microelectrodes to measure changes in ECS, [K+ ]o and [H+ ]o /pHo during electrical stimulation of rat hippocampal slices. Transporters and receptors responding directly to the K+ and glutamate released into the extracellular space (the K+ /Cl- cotransporter, KCC, glutamate transporters and G protein-coupled receptors) did not modulate the extracellular space dynamics. The HCO3--transporting mechanism, which in astrocytes mainly constitutes the electrogenic Na+ / HCO3- cotransporter 1 (NBCe1), is activated by the K+ -mediated depolarization of the astrocytic membrane. Inhibition of this transporter reduced the ECS shrinkage by â¼25% without affecting the K+ transients, pointing to NBCe1 as a key contributor to the stimulus-induced astrocytic cell swelling. Inhibition of the monocarboxylate cotransporters (MCT), like-wise, reduced the ECS shrinkage by â¼25% without compromising the K+ transients. Isosmotic reduction of extracellular Cl- revealed a requirement for this ion in parts of the ECS shrinkage. Taken together, the stimulus-evoked astrocytic cell swelling does not appear to occur as a direct effect of the K+ clearance, as earlier proposed, but partly via the pH-regulating transport mechanisms activated by the K+ -induced astrocytic depolarization and the activity-dependent metabolism.