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The energy cost of information processing is thought to be chiefly neuronal, with a minor fraction attributed to glial cells. However, there is compelling evidence that astrocytes capture synaptic K+ using their Na+/K+ ATPase, and not solely through Kir4.1 channels as was once thought. When this active buffering is taken into account, the cost of astrocytes rises by >200%. Gram-per-gram, astrocytes turn out to be as expensive as neurons. This conclusion is supported by 3D reconstruction of the neuropil showing similar mitochondrial densities in neurons and astrocytes, by cell-specific transcriptomics and proteomics, and by the rates of the tricarboxylic acid cycle. Possible consequences for reactive astrogliosis and brain disease are discussed.
Assuntos
Astrócitos , Encefalopatias , Astrócitos/metabolismo , Humanos , Neuroglia , Neurônios/metabolismo , Sódio/metabolismoRESUMO
BACKGROUND: In the search for new antidepressants, clinical researchers have been using drugs that simultaneously modulate multiple targets. During preclinical and clinical trials, the glutamatergic modulators riluzole and ketamine have received particular attention. Glutamatergic agents have a modulatory effect on synaptic transmission, so they can act on both neurons and astrocytes. In addition to influencing the quantity of glutamate released, these modulators can also affect the expression, localization, and functionality of glutamate-binding sites. OBJECTIVE: This review discusses the complexity of the glutamatergic system, the ambiguity of data regarding glutamate levels in patients with depression, as well as the mechanisms of action for riluzole and ketamine, which includes their relation to the physiology of glutamatergic transmission. The principal aim is to contribute to the development of novel glutamatergic antidepressant medications whilst emphasizing the need for innovative approaches that evaluate their effects on extracellular glutamate. METHODS: Literature was obtained via PubMed by searching the term depression in combination with each of the following terms: riluzole, ketamine, and glutamate. The search was restricted to full-text articles published in English between 1985 and 2018 relating to both the modulatory mechanisms of glutamatergic-binding proteins and the antidepressant actions of these medicines. Articles about mechanisms associated with synaptic plasticity and antidepressant effects were excluded. RESULTS: Although experimental data relates glutamatergic signaling to the pathophysiology of major depression and bipolar disorder, the role of glutamate-as well as its extracellular concentration in patients with said disorders-is still unclear. Riluzole's antidepressant action is ascribed to its capacity to reduce glutamate levels in the synaptic cleft, and ketamine's effect has been associated with increased extracellular glutamate levels. CONCLUSIONS: The strategy of using glutamatergic modulators as therapeutic agents requires a better understanding of the role of glutamate in the pathophysiology of depression. Gaining such understanding is a challenge because it entails evaluating different targets as well as the effects of these modulators on the kinetics of glutamate uptake. Essentially, glutamate transport is a dynamic process and, currently, it is still necessary to develop new approaches to assay glutamate in the synaptic cleft. ORCID: 0000-0002-3358-6939.
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Multiple sclerosis (MS) is a high prevalence degenerative disease characterized at the cellular level by glial and neuronal cell death. The causes of cell death during the disease course are not fully understood. In this work we demonstrate that in a MS model induced by Theiler's murine encephalomyelitis virus (TMEV) infection, the inward rectifier (Kir) 4.1 potassium channel subunit is overexpressed in astrocytes. In voltage clamp experiments the inward current density from TMEV-infected astrocytes was significantly larger than in mock-infected ones. The cRNA hybridization analysis from mock- and TMEV-infected cells showed an upregulation of a potassium transport channel coding sequence. We validated this mRNA increase by RT-PCR and quantitative PCR using Kir 4.1 specific primers. Western blotting experiments confirmed the upregulation of Kir 4.1, and alignment between sequences provided the demonstration that the over-expressed gene encodes for a Kir family member. Flow cytometry showed that the Kir 4.1 protein is located mainly in the cell membrane in mock and TMEV-infected astrocytes. Our results demonstrate an increase in K+ inward current in TMEV-infected glial cells, this increment may reduce the neuronal depolarization, contributing to cell resilience mechanisms.
Assuntos
Astrócitos/metabolismo , Esclerose Múltipla/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Infecções por Cardiovirus/complicações , Infecções por Cardiovirus/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Potenciais da Membrana , Mesocricetus , Esclerose Múltipla/virologia , RNA Mensageiro , Theilovirus/patogenicidade , Regulação para CimaRESUMO
Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.
Assuntos
Pentamidina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Sítios de Ligação , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Pentamidina/metabolismo , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
Sertraline (Zoloft) and fluoxetine (Prozac) are selective serotonin reuptake inhibitors whose antidepressant mechanism of action is classically attributed to an elevation of the extracellular levels of serotonin in the synaptic cleft. However, the biological effects of these drugs seem to be more complex than their traditionally described mechanism of action. Among their actions is the inhibition of different types of Na+ and K+ channels, as well as of glutamate uptake activity. The clearance of extracellular glutamate is essential to maintain the central nervous system within physiological conditions, and this excitatory neurotransmitter is removed from the synaptic cleft by astrocyte transporters. This transport depends upon a hyperpolarized membrane potential in astrocytes that is mainly maintained by Kir4.1 K+ channels. The impairment of the Kir4.1 channel activity reduces driving force for the glutamate transporter, resulting in an accumulation of extracellular glutamate. It has been shown that sertraline and fluoxetine inhibit Kir4.1 K+ channels. Recently, we demonstrated that sertraline reduces glutamate uptake in human platelets, which contain a high-affinity Na+-dependent glutamate uptake system, with kinetic and pharmacological properties similar to astrocytes in the central nervous system. Considering these similarities between human platelets and astrocytes, one might ask if sertraline could potentially reduce glutamate clearance in the synaptic cleft and consequently modulate glutamatergic transmission. This possibility merits investigation, since it may provide additional information regarding the mechanism of action and perhaps the side effects of these antidepressants.
RESUMO
Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3µM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Quinacrina/farmacologia , Animais , Astrócitos/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp/métodos , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Quinacrina/metabolismo , RatosRESUMO
Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.