RESUMO
This study demonstrates that the action potential discharge in vagal afferent A-fiber neurons is about 20 times more sensitive to the rate of membrane depolarization compared to C-fiber neurons. The sensitivity of action potential generation to the depolarization rate in vagal sensory neurons is independent of the intensity of current stimuli but nearly abrogated by inhibiting the D-type potassium channel. These findings help better understand the mechanisms that control the activation of vagal afferent nerves.
Assuntos
Potenciais de Ação/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Fibras Nervosas Amielínicas/fisiologia , Neurônios Aferentes/fisiologia , Gânglio Nodoso/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidoresRESUMO
Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.
Assuntos
Envelhecimento , Proteínas de Drosophila/fisiologia , Drosophila , Coração/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Animais , Arritmias Cardíacas/genética , Técnicas de Silenciamento de Genes , Insuficiência Cardíaca/genéticaRESUMO
Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K+ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.
Assuntos
Aciltransferases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Habituação Psicofisiológica/genética , Aprendizagem/fisiologia , Lipoilação/genética , Lipoilação/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Terminações Pré-Sinápticas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
Despite major advances in the structure determination of ion channels, the sequence of molecular rearrangements at negative membrane potentials in voltage-gated potassium channels of the Shaker family remains unknown. Four major composite gating states are documented during the gating process: closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI). Although many steps in the gating cycle have been clarified experimentally, the development of steady-state inactivation at negative membrane potentials and mandatory gating transitions for recovery from inactivation have not been elucidated. In this study, we exploit the biophysical properties of Shaker-IR mutants T449A/V474C and T449A/V476C to evaluate the status of the activation and inactivation gates during steady-state inactivation and upon locking the channel open with intracellular Cd2+. We conclude that at negative membrane potentials, the gating scheme of Shaker channels can be refined in two aspects. First, the most likely pathway for the development of steady-state inactivation is CâOâOIâCI. Second, the OIâCI transition is a prerequisite for recovery from inactivation. These findings are in accordance with the widely accepted view that tight coupling is present between the activation and C-type inactivation gates in Shaker and underscore the role of steady-state inactivation and recovery from inactivation as determinants of excitability.
Assuntos
Ativação do Canal Iônico , Potenciais da Membrana , Superfamília Shaker de Canais de Potássio/fisiologia , CinéticaRESUMO
The model legume Medicago truncatula possesses a single outward Shaker K+ channel, whereas Arabidopsis thaliana possesses two channels of this type, named AtSKOR and AtGORK, with AtSKOR having been shown to play a major role in K+ secretion into the xylem sap in the root vasculature and with AtGORK being shown to mediate the efflux of K+ across the guard cell membrane, leading to stomatal closure. Here we show that the expression pattern of the single M. truncatula outward Shaker channel, which has been named MtGORK, includes the root vasculature, guard cells and root hairs. As shown by patch-clamp experiments on root hair protoplasts, besides the Shaker-type slowly activating outwardly rectifying K+ conductance encoded by MtGORK, a second K+ -permeable conductance, displaying fast activation and weak rectification, can be expressed by M. truncatula. A knock-out (KO) mutation resulting in an absence of MtGORK activity is shown to weakly reduce K+ translocation to shoots, and only in plants engaged in rhizobial symbiosis, but to strongly affect the control of stomatal aperture and transpirational water loss. In legumes, the early electrical signaling pathway triggered by Nod-factor perception is known to comprise a short transient depolarization of the root hair plasma membrane. In the absence of the functional expression of MtGORK, the rate of the membrane repolarization is found to be decreased by a factor of approximately two. This defect was without any consequence on infection thread development and nodule production in plants grown in vitro, but a decrease in nodule production was observed in plants grown in soil.
Assuntos
Medicago truncatula/metabolismo , Proteínas de Plantas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Técnicas de Inativação de Genes , Medicago truncatula/genética , Medicago truncatula/fisiologia , Oócitos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Transpiração Vegetal , Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/fisiologia , XenopusRESUMO
MAIN CONCLUSION: PbrKAT1, which is inhibited by external Na+ in Xenopus laevis oocytes, is characterized as encoding a typical inward rectifying channel that is mainly expressed in guard cells. Potassium (K+) is the most abundant cation in plant cells necessary for plant growth and development. The uptake and transport of K+ are mainly completed through transporters and channels, and the Shaker family genes are the most studied K+ channels in plants. However, there is far less information about this family in Rosaceae species. We performed a genome-wide analysis and identified Shaker K+ channel gene family members in Rosaceae. We cloned and characterized a Shaker K+ channel KAT1 from pear (Pyrus × bretschneideri). In total, 36 Shaker K+ channel genes were identified from Rosaceae species and were classified into five subgroups based on structural characteristics and a phylogenetic analysis. Whole-genome and dispersed duplications were the primary forces underlying Shaker K+ channel gene family expansion in Rosaceae, and purifying selection played a key role in the evolution of Shaker K+ channel genes. ß-Glucuronidase and qRT-PCR assays revealed that PbrKAT1 was mainly expressed in leaves, especially in guard cells. PbrKAT1 displayed a typical inward-rectifying current when expressed in Xenopus laevis oocytes. The activity of PbrKAT1 was inhibited by external sodium ions, possibly playing an important role in the regulation of salt tolerance in pear. These results provide valuable information on evolution, expression and functions of the Shaker K+ channel gene family in plants.
Assuntos
Proteínas de Plantas/metabolismo , Pyrus/metabolismo , Rosaceae/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Animais Geneticamente Modificados , Arabidopsis/genética , Arabidopsis/metabolismo , Cromossomos de Plantas/genética , Clonagem Molecular , Evolução Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Filogenia , Proteínas de Plantas/fisiologia , Reação em Cadeia da Polimerase , Pyrus/fisiologia , Rosaceae/genética , Rosaceae/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Sintenia/genética , Xenopus laevisRESUMO
During early postnatal life, speed up of signal propagation through many central and peripheral neurons has been associated with an increase in axon diameter or/and myelination. Especially in unmyelinated axons postnatal adjustments of axonal membrane conductances is potentially a third mechanism but solid evidence is lacking. Here, we show that axonal action potential (AP) conduction velocity in the Drosophila giant fiber (GF) interneuron, which is required for fast long-distance signal conduction through the escape circuit, is increased by 80% during the first day of adult life. Genetic manipulations indicate that this postnatal increase in AP conduction velocity in the unmyelinated GF axon is likely owed to adjustments of ion channel expression or properties rather than axon diameter increases. Specifically, targeted RNAi knock-down of either Para fast voltage-gated sodium, Shaker potassium (Kv1 homologue), or surprisingly, L-type like calcium channels counteracts postnatal increases in GF axonal conduction velocity. By contrast, the calcium-dependent potassium channel Slowpoke (BK) is not essential for postnatal speeding, although it also significantly increases conduction velocity. Therefore, we identified multiple ion channels that function to support fast axonal AP conduction velocity, but only a subset of these are regulated during early postnatal life to maximize conduction velocity. Despite its large diameter (â¼7 µm) and postnatal regulation of multiple ionic conductances, mature GF axonal conduction velocity is still 20-60 times slower than that of vertebrate Aß sensory axons and α motoneurons, thus unraveling the limits of long-range information transfer speed through invertebrate circuits.
Assuntos
Axônios/fisiologia , Canais de Cálcio Tipo L/fisiologia , Drosophila/fisiologia , Interneurônios/fisiologia , Condução Nervosa/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologia , Potenciais de Ação/fisiologia , Animais , Drosophila/crescimento & desenvolvimento , Feminino , Larva/crescimento & desenvolvimento , Larva/fisiologia , MasculinoRESUMO
Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma-1 receptor (Sig-1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig-1R and Kv1.2 has not been elucidated. We hypothesized that Sig-1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK293 cells, we found that ligand activation of the Sig-1R modulates Kv1.2 current amplitude. More importantly, Sig-1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kvß2 and when the Sig-1R mutation underlying ALS16 (Sig-1R-E102Q), is expressed. These data suggest that Kvß2 occludes the interaction of Sig-1R with Kv1.2, and that E102 may be a residue critical for Sig-1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig-1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig-1R dysfunction.
Assuntos
Canal de Potássio Kv1.2/fisiologia , Receptores sigma/fisiologia , Células Cultivadas , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.2/efeitos dos fármacos , Canal de Potássio Kv1.2/metabolismo , Técnicas de Patch-Clamp/métodos , Fenazocina/análogos & derivados , Fenazocina/antagonistas & inibidores , Fenazocina/farmacologia , Receptores sigma/agonistas , Receptores sigma/metabolismo , Superfamília Shaker de Canais de Potássio/fisiologia , Receptor Sigma-1RESUMO
We used voltage-clamp recordings from somatic outside-out macropatches to determine the amplitude and biophysical properties of putative Kv1-mediated currents in layer 5 pyramidal neurons (PNs) from mice expressing EGFP under the control of promoters for etv1 or glt. We then used whole cell current-clamp recordings and Kv1-specific peptide blockers to test the hypothesis that Kv1 channels differentially regulate action potential (AP) voltage threshold, repolarization rate, and width as well as rheobase and repetitive firing in these two PN types. We found that Kv1-mediated currents make up a similar percentage of whole cell K+ current in both cell types, and only minor biophysical differences were observed between PN types or between currents sensitive to different Kv1 blockers. Putative Kv1 currents contributed to AP voltage threshold in both PN types, but AP width and rate of repolarization were only affected in etv1 PNs. Kv1 currents regulate rheobase, delay to the first AP, and firing rate similarly in both cell types, but the frequency-current slope was much more sensitive to Kv1 block in etv1 PNs. In both cell types, Kv1 block shifted the current required to elicit an onset doublet of action potentials to lower currents. Spike frequency adaptation was also affected differently by Kv1 block in the two PN types. Thus, despite similar expression levels and minimal differences in biophysical properties, Kv1 channels differentially regulate APs and repetitive firing in etv1 and glt PNs. This may reflect differences in subcellular localization of channel subtypes or differences in the other K+ channels expressed. NEW & NOTEWORTHY In two types of genetically identified layer 5 pyramidal neurons, α-dendrotoxin blocked approximately all of the putative Kv1 current (on average). We used outside-out macropatches and whole cell recordings at 33°C to show that despite similar expression levels and minimal differences in biophysical properties, Kv1 channels differentially regulate action potentials and repetitive firing in etv1 and glt pyramidal neurons. This may reflect differences in subcellular localization of channel subtypes or differences in the other K+ channels expressed.
Assuntos
Proteínas de Ligação a DNA/genética , Células Piramidais/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Córtex Somatossensorial/fisiologia , Fatores de Transcrição/genética , Potenciais de Ação , Animais , Feminino , Masculino , Camundongos , Células Piramidais/metabolismo , Córtex Somatossensorial/metabolismoRESUMO
Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.
Assuntos
Gânglios Espinais/fisiopatologia , Imunoglobulina G/administração & dosagem , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Dor Nociceptiva/imunologia , Dor Nociceptiva/fisiopatologia , Células Receptoras Sensoriais/fisiologia , Animais , Células Cultivadas , Feminino , Humanos , Imunização Passiva , Masculino , Mecanotransdução Celular , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Células do Corno Posterior/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologiaRESUMO
In the last years it has been increasingly clear that KV-channel activity modulates neurotransmitter release. The subcellular localization and composition of potassium channels are crucial to understanding its influence on neurotransmitter release. To investigate the role of KV in corticostriatal synapses modulation, we combined extracellular recording of population-spike and pharmacological blockage with specific and nonspecific blockers to identify several families of KV channels. We induced paired-pulse facilitation (PPF) and studied the changes in paired-pulse ratio (PPR) before and after the addition of specific KV blockers to determine whether particular KV subtypes were located pre- or postsynaptically. Initially, the presence of KV channels was tested by exposing brain slices to tetraethylammonium or 4-aminopyridine; in both cases we observed a decrease in PPR that was dose dependent. Further experiments with tityustoxin, margatoxin, hongotoxin, agitoxin, dendrotoxin, and BDS-I toxins all rendered a reduction in PPR. In contrast heteropodatoxin and phrixotoxin had no effect. Our results reveal that corticostriatal presynaptic KV channels have a complex stoichiometry, including heterologous combinations KV1.1, KV1.2, KV1.3, and KV1.6 isoforms, as well as KV3.4, but not KV4 channels. The variety of KV channels offers a wide spectrum of possibilities to regulate neurotransmitter release, providing fine-tuning mechanisms to modulate synaptic strength.
Assuntos
Córtex Cerebral/fisiologia , Corpo Estriado/fisiologia , Terminações Pré-Sinápticas/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Canais de Potássio Shaw/fisiologia , Sinapses/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Masculino , Bloqueadores dos Canais de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/fisiologia , Ratos , Ratos Wistar , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Canais de Potássio Shaw/antagonistas & inibidores , Sinapses/efeitos dos fármacosRESUMO
Neuronal subtype-specific transcription factors (TFs) instruct key features of neuronal function and connectivity. Activity-dependent mechanisms also contribute to wiring and circuit assembly, but whether and how they relate to TF-directed neuronal differentiation is poorly investigated. Here we demonstrate that the TF Cux1 controls the formation of the layer II/III corpus callosum (CC) projections through the developmental transcriptional regulation of Kv1 voltage-dependent potassium channels and the resulting postnatal switch to a Kv1-dependent firing mode. Loss of Cux1 function led to a decrease in the expression of Kv1 transcripts, aberrant firing responses, and selective loss of CC contralateral innervation. Firing and innervation were rescued by re-expression of Kv1 or postnatal reactivation of Cux1. Knocking down Kv1 mimicked Cux1-mediated CC axonal loss. These findings reveal that activity-dependent processes are central bona fide components of neuronal TF-differentiation programs and establish the importance of intrinsic firing modes in circuit assembly within the neocortex.
Assuntos
Potenciais de Ação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Superfamília Shaker de Canais de Potássio/fisiologia , Animais , Corpo Caloso/citologia , Corpo Caloso/crescimento & desenvolvimento , Corpo Caloso/fisiologia , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Cultura Primária de Células , Superfamília Shaker de Canais de Potássio/biossíntese , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
The discrete arrangement of voltage-gated K(+) (Kv) channels in axons may impart functional advantages in action potential (AP) signaling yet, in compact cell types, the organization of Kv channels is poorly understood. We find that in cerebellar stellate cell interneurons of mice, the composition and influence of Kv channels populating the axon is diverse and depends on location allowing axonal compartments to differentially control APs in a local manner. Kv1 channels determine AP repolarization at the spike initiation site but not at more distal sites, limiting the expression of use-dependent spike broadening to the most proximal axon region, likely a key attribute informing spiking phenotype. Local control of AP repolarization at presynaptic boutons depends on Kv3 channels keeping APs brief, thus limiting Ca(2+) influx and synaptic strength. These observations suggest that AP repolarization is tuned by the local influence of distinct Kv channel types, and this organization enhances the functional segregation of axonal compartments.
Assuntos
Axônios/fisiologia , Cerebelo/fisiologia , Interneurônios/fisiologia , Canais de Potássio/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Pré-Sinápticos/fisiologia , Animais , Cerebelo/citologia , Fenômenos Eletrofisiológicos , Feminino , Corantes Fluorescentes , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Receptores de AMPA/fisiologia , Receptores de GABA-A/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Canais de Potássio Shaw/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.
Assuntos
Artérias Cerebrais/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Masculino , Modelos Animais , Técnicas de Patch-Clamp , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
BACKGROUND: Although sleep is conserved throughout evolution, the molecular basis of its control is still largely a mystery. We previously showed that the quiver/sleepless (qvr/sss) gene encodes a membrane-tethered protein that is required for normal sleep in Drosophila. SLEEPLESS (SSS) protein functions, at least in part, by upregulating the levels and open probability of Shaker (Sh) potassium channels to suppress neuronal excitability and enable sleep. Consistent with this proposed mechanism, loss-of-function mutations in Sh phenocopy qvr/sss-null mutants. However, sleep is more genetically modifiable in Sh than in qvr/sss mutants, suggesting that SSS may regulate additional molecules to influence sleep. RESULTS: Here we show that SSS also antagonizes nicotinic acetylcholine receptors (nAChRs) to reduce synaptic transmission and promote sleep. Mimicking this antagonism with the nAChR inhibitor mecamylamine or by RNAi knockdown of specific nAChR subunits is sufficient to restore sleep to qvr/sss mutants. Regulation of nAChR activity by SSS occurs posttranscriptionally, since the levels of nAChR mRNAs are unchanged in qvr/sss mutants. Regulation of nAChR activity by SSS may in fact be direct, since SSS forms a stable complex with and antagonizes nAChR function in transfected cells. Intriguingly, lynx1, a mammalian homolog of SSS, can partially restore normal sleep to qvr/sss mutants, and lynx1 can form stable complexes with Shaker-type channels and nAChRs. CONCLUSIONS: Together, our data point to an evolutionarily conserved, bifunctional role for SSS and its homologs in controlling excitability and synaptic transmission in fundamental processes of the nervous system such as sleep.
Assuntos
Neurônios Colinérgicos/fisiologia , Proteínas de Drosophila/fisiologia , Proteínas de Membrana/fisiologia , Sono/fisiologia , Transmissão Sináptica/fisiologia , Animais , Drosophila melanogaster , Técnicas de Silenciamento de Genes , Corpos Pedunculados/fisiologia , Receptores Nicotínicos/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Vigília/fisiologiaRESUMO
Polyunsaturated fatty acids modulate the voltage dependence of several voltage-gated ion channels, thereby being potent modifiers of cellular excitability. Detailed knowledge of this molecular mechanism can be used in designing a new class of small-molecule compounds against hyperexcitability diseases. Here, we show that arginines on one side of the helical K-channel voltage sensor S4 increased the sensitivity to docosahexaenoic acid (DHA), whereas arginines on the opposing side decreased this sensitivity. Glutamates had opposite effects. In addition, a positively charged DHA-like molecule, arachidonyl amine, had opposite effects to the negatively charged DHA. This suggests that S4 rotates to open the channel and that DHA electrostatically affects this rotation. A channel with arginines in positions 356, 359, and 362 was extremely sensitive to DHA: 70 µM DHA at pH 9.0 increased the current >500 times at negative voltages compared with wild type (WT). The small-molecule compound pimaric acid, a novel Shaker channel opener, opened the WT channel. The 356R/359R/362R channel drastically increased this effect, suggesting it to be instrumental in future drug screening.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Superfamília Shaker de Canais de Potássio/agonistas , Superfamília Shaker de Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Feminino , Canais Iônicos/química , Canais Iônicos/fisiologia , Dados de Sequência Molecular , Mutação/fisiologia , Superfamília Shaker de Canais de Potássio/química , Xenopus laevisRESUMO
Axons degenerate after injury and in neuropathies and disease via a self-destruction program whose mechanism is poorly understood. Axons that have lost connection to their cell bodies have altered electrical and synaptic activities, but whether such changes play a role in the axonal degeneration process is not clear. We have used a Drosophila model to study the Wallerian degeneration of motoneuron axons and their neuromuscular junction synapses. We found that degeneration of the distal nerve stump after a nerve crush is greatly delayed when there is increased potassium channel activity (by overexpression of two different potassium channels, Kir2.1 and dORKΔ-C) or decreased voltage-gated sodium channel activity (using mutations in the para sodium channel). Conversely, degeneration is accelerated when potassium channel activity is decreased (by expressing a dominant-negative mutation of Shaker). Despite the effect of altering voltage-gated sodium and potassium channel activity, recordings made after nerve crush demonstrated that the distal stump does not fire action potentials. Rather, a variety of lines of evidence suggest that the sodium and potassium channels manifest their effects upon degeneration through changes in the resting membrane potential, which in turn regulates the level of intracellular free calcium within the isolated distal axon.
Assuntos
Axônios/fisiologia , Drosophila/fisiologia , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia , Degeneração Walleriana/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Cálcio/metabolismo , Fenômenos Eletrofisiológicos/fisiologia , Imuno-Histoquímica , Microscopia Confocal , Compressão Nervosa , Junção Neuromuscular/fisiologia , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/fisiologia , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/fisiologia , Temperatura , Tetrodotoxina/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Heterozygous mutations in KCNA1 cause episodic ataxia type 1 (EA1), an ion channel disorder characterised by brief paroxysms of cerebellar dysfunction and persistent neuromyotonia. This paper describes four previously unreported families with EA1, with the aim of understanding the phenotypic spectrum associated with different mutations. METHODS: 15 affected individuals from four families underwent clinical, genetic and neurophysiological evaluation. The functional impact of new mutations identified in the KCNA1 gene was investigated with in vitro electrophysiology and immunocytochemistry. RESULTS: Detailed clinical documentation, dating back to 1928 in one family, indicates that all patients manifested episodic ataxia of varying severity. Four subjects from three families reported hearing impairment, which has not previously been reported in association with EA1. New mutations (R167M, C185W and I407M) were identified in three out of the four families. When expressed in human embryonic kidney cells, all three new mutations resulted in a loss of K(v)1.1 channel function. The fourth family harboured a previously reported A242P mutation, which has not been previously described in association with ataxia. CONCLUSIONS: The genetic basis of EA1 in four families is established and this report presents the earliest documented case from 1928. All three new mutations caused a loss of K(v)1.1 channel function. The finding of deafness in four individuals raises the possibility of a link between K(v)1.1 dysfunction and hearing impairment. Our findings broaden the phenotypic range associated with mutations in KCNA1.
Assuntos
Ataxia/diagnóstico , Ataxia/genética , Análise Mutacional de DNA , Triagem de Portadores Genéticos , Canal de Potássio Kv1.1/genética , Mioquimia/diagnóstico , Mioquimia/genética , Adolescente , Ataxia/fisiopatologia , Linhagem Celular Transformada , Cerebelo/fisiopatologia , Cromossomos Humanos Par 12/genética , Avaliação da Deficiência , Eletromiografia , Feminino , Humanos , Técnicas In Vitro , Síndrome de Isaacs/diagnóstico , Síndrome de Isaacs/genética , Síndrome de Isaacs/fisiopatologia , Masculino , Neurônios Motores/fisiologia , Mioquimia/fisiopatologia , Linhagem , Fenótipo , Análise de Sequência de DNA , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/fisiologia , TransfecçãoRESUMO
The output of the cerebellum to the motor axis of the central nervous system is orchestrated mainly by synaptic inputs and intrinsic pacemaker activity of deep cerebellar nuclear (DCN) projection neurons. Herein, we demonstrate that the soma of these cells is enriched with K(V)1 channels produced by mandatory multi-merization of K(V)1.1, 1.2 α and KV ß2 subunits. Being constitutively active, the K(+) current (IK(V)1) mediated by these channels stabilizes the rate and regulates the temporal precision of self-sustained firing of these neurons. Placed strategically, IK(V)1 provides a powerful counter-balance to prolonged depolarizing inputs, attenuates the rebound excitation, and dampens the membrane potential bi-stability. Somatic location with low activation threshold render IK(V)1 instrumental in voltage-dependent de-coupling of the axon initial segment from the cell body of projection neurons, impeding invasion of back-propagating action potentials into the somato-dendritic compartment. The latter is also demonstrated to secure the dominance of clock-like somatic pacemaking in driving the regenerative firing activity of these neurons, to encode time variant inputs with high fidelity. Through the use of multi-compartmental modelling and retro-axonal labelling, the physiological significance of the described functions for processing and communication of information from the lateral DCN to thalamic relay nuclei is established.
Assuntos
Núcleos Cerebelares/fisiologia , Neurônios/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Tálamo/fisiologia , Animais , Relógios Biológicos , Núcleos Cerebelares/citologia , Técnicas In Vitro , Subunidades Proteicas/fisiologia , RatosRESUMO
Voltage-activated ion channels open and close in response to changes in membrane voltage, a property that is fundamental to the roles of these channels in electrical signaling. Protein toxins from venomous organisms commonly target the S1-S4 voltage-sensing domains in these channels and modify their gating properties. Studies on the interaction of hanatoxin with the Kv2.1 channel show that this tarantula toxin interacts with the S1-S4 domain and inhibits opening by stabilizing a closed state. Here we investigated the interaction of hanatoxin with the Shaker Kv channel, a voltage-activated channel that has been extensively studied with biophysical approaches. In contrast to what is observed in the Kv2.1 channel, we find that hanatoxin shifts the conductance-voltage relation to negative voltages, making it easier to open the channel with membrane depolarization. Although these actions of the toxin are subtle in the wild-type channel, strengthening the toxin-channel interaction with mutations in the S3b helix of the S1-S4 domain enhances toxin affinity and causes large shifts in the conductance-voltage relationship. Using a range of previously characterized mutants of the Shaker Kv channel, we find that hanatoxin stabilizes an activated conformation of the voltage sensors, in addition to promoting opening through an effect on the final opening transition. Chimeras in which S3b-S4 paddle motifs are transferred between Kv2.1 and Shaker Kv channels, as well as experiments with the related tarantula toxin GxTx-1E, lead us to conclude that the actions of tarantula toxins are not simply a product of where they bind to the channel, but that fine structural details of the toxin-channel interface determine whether a toxin is an inhibitor or opener.