RESUMO
Dynamic microtubules critically regulate synaptic functions, but the role of microtubule severing in these processes is barely understood. Katanin is a neuronally expressed microtubule-severing complex regulating microtubule number and length in cell division or neurogenesis; however, its potential role in synaptic functions has remained unknown. Studying mice from both sexes, we found that katanin is abundant in neuronal dendrites and can be detected at individual excitatory spine synapses. Overexpression of a dominant-negative ATPase-deficient katanin subunit to functionally inhibit severing alters the growth of microtubules in dendrites, specifically at premature but not mature neuronal stages without affecting spine density. Notably, interference with katanin function prevented structural spine remodeling following single synapse glutamate uncaging and significantly affected the potentiation of AMPA-receptor-mediated excitatory currents after chemical induction of long-term potentiation. Furthermore, katanin inhibition reduced the invasion of microtubules into fully developed spines. Our data demonstrate that katanin-mediated microtubule severing regulates structural and functional plasticity at synaptic sites.
Assuntos
Microtúbulos , Neurônios , Animais , Camundongos , Katanina/genética , Katanina/metabolismo , Microtúbulos/metabolismo , Neurônios/fisiologia , Neurogênese , Plasticidade NeuronalRESUMO
Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.
Assuntos
Ácido Glutâmico/metabolismo , Cinesinas/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismoRESUMO
In myelinated nerve fibres, action potentials are generated at nodes of Ranvier. These structures are located at interruptions of the myelin sheath, forming narrow gaps with small rings of axolemma freely exposed to the extracellular space. The mammalian node contains a high density of Na+ channels and K+ -selective leakage channels. Voltage-dependent Kv1 channels are only present in the juxta-paranode. Recently, the leakage channels have been identified as K2P channels (TRAAK, TREK-1). K2P channels are K+ -selective 'background' channels, characterized by outward rectification and their ability to be activated, e.g. by temperature, mechanical stretch or arachidonic acid. We are only beginning to elucidate the peculiar functions of nodal K2P channels. I will discuss two functions of the nodal K2P-mediated conductance. First, at body temperature K2P channels have a high open probability, thereby inducing a resting potential of about -85 mV. This negative resting potential reduces steady-state Na+ channel inactivation and ensures a large Na+ inward current upon a depolarizing stimulus. Second, the K2P conductance is involved in nodal action potential repolarization. The identification of nodal K2P channels is exciting since it shows that the nodal K+ conductance is not a fixed value but can be changed: it can be increased or decreased by a broad range of K2P modulators, thereby modulating, for example, the resting potential. The functional importance of nodal K2P channels will be exemplified by describing in more detail the function of the K2P conductance increase by raising the temperature from room temperature to 37°C.
Assuntos
Axônios , Fibras Nervosas Mielinizadas , Potenciais de Ação , Animais , Potenciais da Membrana , Bainha de MielinaRESUMO
Mammalian ether-à-go-go (EAG) channels are voltage-gated K+ channels. They are encoded by the KCNH gene family and divided into three subfamilies, eag (Kv10), erg (eag-related gene; Kv11) and elk (eag-like; Kv12). All EAG channel subtypes are expressed in the brain where they effectively modulate neuronal excitability. This Topical Review describes the biophysical properties of each of the EAG channel subtypes, their function in neurons and the neurological diseases induced by EAG channel mutations. In contrast to the function of erg currents in the heart, where they contribute to repolarization of the cardiac action potential, erg currents in neurons are involved in the maintenance of the resting potential, setting of action potential threshold and frequency accommodation. They can even support high frequency firing by preventing a depolarization-induced Na+ channel block. EAG channels are modulated differentially, e.g. eag channels by intracellular Ca2+ , erg channels by extracellular K+ and GPCRs, and elk channels by changes in pH. So far, only currents mediated by erg channels have been recorded in neurons with the help of selective blockers. Neuronal eag and elk currents have not been isolated due to the lack of suitable channel blockers. However, findings in KO mice indicate a physiological role of eag1 currents in synaptic transmission and an involvement of elk2 currents in cognitive performance. Human eag1 and eag2 gain-of-function mutations underlie syndromes associated with epileptic seizures.
Assuntos
Potenciais de Ação , Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais da Membrana , Neurônios/fisiologia , Animais , HumanosRESUMO
GABAA receptors shape synaptic transmission by modulating Cl(-) conductance across the cell membrane. Remarkably, animal toxins that specifically target GABAA receptors have not been identified. Here, we report the discovery of micrurotoxin1 (MmTX1) and MmTX2, two toxins present in Costa Rican coral snake venom that tightly bind to GABAA receptors at subnanomolar concentrations. Studies with recombinant and synthetic toxin variants on hippocampal neurons and cells expressing common receptor compositions suggest that MmTX1 and MmTX2 allosterically increase GABAA receptor susceptibility to agonist, thereby potentiating receptor opening as well as desensitization, possibly by interacting with the α(+)/ß(-) interface. Moreover, hippocampal neuron excitability measurements reveal toxin-induced transitory network inhibition, followed by an increase in spontaneous activity. In concert, toxin injections into mouse brain result in reduced basal activity between intense seizures. Altogether, we characterized two animal toxins that enhance GABAA receptor sensitivity to agonist, thereby establishing a previously unidentified class of tools to study this receptor family.
Assuntos
Venenos Elapídicos/farmacologia , Elapidae/metabolismo , Peptídeos/farmacologia , Receptores de GABA-A/metabolismo , Sequência de Aminoácidos , Animais , Venenos Elapídicos/química , Células HEK293 , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Masculino , Camundongos , Dados de Sequência Molecular , Mutação/genética , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores de GABA-A/química , Receptores de GABA-A/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , XenopusRESUMO
Since the first discovery of Kvbeta-subunits more than 15 years ago, many more ancillary Kv channel subunits were characterized, for example, KChIPs, KCNEs, and BKbeta-subunits. The ancillary subunits are often integral parts of native Kv channels, which, therefore, are mostly multiprotein complexes composed of voltage-sensing and pore-forming Kvalpha-subunits and of ancillary or beta-subunits. Apparently, Kv channels need the ancillary subunits to fulfill their many different cell physiological roles. This is reflected by the large structural diversity observed with ancillary subunit structures. They range from proteins with transmembrane segments and extracellular domains to purely cytoplasmic proteins. Ancillary subunits modulate Kv channel gating but can also have a great impact on channel assembly, on channel trafficking to and from the cellular surface, and on targeting Kv channels to different cellular compartments. The importance of the role of accessory subunits is further emphasized by the number of mutations that are associated in both humans and animals with diseases like hypertension, epilepsy, arrhythmogenesis, periodic paralysis, and hypothyroidism. Interestingly, several ancillary subunits have in vitro enzymatic activity; for example, Kvbeta-subunits are oxidoreductases, or modulate enzymatic activity, i.e., KChIP3 modulates presenilin activity. Thus different modes of beta-subunit association and of functional impact on Kv channels can be delineated, making it difficult to extract common principles underlying Kvalpha- and beta-subunit interactions. We critically review present knowledge on the physiological role of ancillary Kv channel subunits and their effects on Kv channel properties.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Humanos , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Subunidades ProteicasRESUMO
We investigated the subthreshold properties of an erg (ether-à-go-go-related gene) K(+) current in Purkinje cells of neonatal mice. Action potentials recorded from Purkinje cells in cerebellar slices exhibited a decreased threshold potential and increased frequency of spontaneous and repetitive activity following application of the specific erg channel blocker E-4031. Accommodation was absent before and after drug application. The erg current of these Purkinje cells activated at membrane potentials near -60 mV and exhibited fast gating kinetics. The functional importance of fast gating subthreshold erg channels in Purkinje cells was corroborated by comparing the results of action potential clamp experiments with erg1a, erg1b, erg2, and erg3 currents heterologously expressed in HEK cells. Computer simulations based on a NEURON model of Purkinje cells only reproduced the effects of the native erg current when an erg channel conductance like that of erg3 was included. Experiments with subunit-sensitive toxins (BeKm-1, APETx1) indicated that erg channels in Purkinje cells are presumably mediated by heteromeric erg1/erg3 or modified erg1 channels. Following mGluR1 activation, the native erg current was reduced by â¼70%, brought about by reduction of the maximal erg current and a shift of the activation curve to more positive potentials. The Purkinje cell erg current contributed to the sustained current component of the biphasic mGluR1 response. Activation of mGluR1 by the agonist 3,4-dihydroxyphenylglycol increased Purkinje cell excitability, similar to that induced by E-4031. The results indicated that erg currents can be modulated and may contribute to the mGluR1-induced plasticity changes in Purkinje cells.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Potenciais da Membrana/fisiologia , Células de Purkinje/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Potenciais de Ação/fisiologia , Animais , Cerebelo/metabolismo , Venenos de Cnidários/farmacologia , Simulação por Computador , Agonistas de Aminoácidos Excitatórios/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Neurológicos , Receptores de Glutamato Metabotrópico/agonistas , Venenos de Escorpião/farmacologia , Sesquiterpenos/farmacologia , Sesquiterpenos de GuaianoRESUMO
KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.
Assuntos
Canal de Potássio KCNQ1 , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Simulação de Dinâmica Molecular , Oócitos/metabolismo , Técnicas de Patch-ClampRESUMO
HERG (human ether-à-go-go-related gene) K(+) currents fulfill important ionic functions in cardiac and other excitable cells. In addition, HERG channels influence cell growth and migration in various types of tumor cells. The mechanisms underlying these functions are still not resolved. Here, we investigated the role of HERG channels for cell growth in a cell line (SW2) derived from small cell lung cancer (SCLC), a malignant variant of lung cancer. The two HERG1 isoforms (HERG1a, HERG1b) as well as HERG2 and HERG3 are expressed in SW2 cells. Inhibition of HERG currents by acute or sustained application of E-4031, a specific ERG channel blocker, depolarized SW2 cells by 10-15 mV. This result indicated that HERG K(+) conductance contributes considerably to the maintenance of the resting potential of about -45 mV. Blockage of HERG channels by E-4031 for up to 72 h did not affect cell proliferation. In contrast, siRNA-induced inhibition of HERG1 protein expression decreased cell proliferation by about 50%. Reduction of HERG1 protein expression was confirmed by Western blots. HERG current was almost absent in SW2 cells transfected with siRNA against HERG1. Qualitatively similar results were obtained in three other SCLC cell lines (OH1, OH3, H82), suggesting that the HERG1 channel protein is involved in SCLC cell growth, whereas the ion-conducting function of HERG1 seems not to be important for cell growth.
Assuntos
Proliferação de Células , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Piperidinas/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation.
Assuntos
Actinas , Extinção Psicológica , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Cognição , Medo , CamundongosRESUMO
Neuroligins are postsynaptic cell adhesion molecules that associate with presynaptic neurexins. Both factors form a transsynaptic connection, mediate signaling across the synapse, specify synaptic functions, and play a role in synapse formation. Neuroligin dysfunction impairs synaptic transmission, disrupts neuronal networks, and is thought to participate in cognitive diseases. Here we report that chemical treatment designed to induce long-term potentiation or long-term depression (LTD) induces neuroligin 1/3 turnover, leading to either increased or decreased surface membrane protein levels, respectively. Despite its structural role at a crucial transsynaptic position, GFP-neuroligin 1 leaves synapses in hippocampal neurons over time with chemical LTD-induced neuroligin internalization depending on an intact microtubule cytoskeleton. Accordingly, neuroligin 1 and its binding partner postsynaptic density protein-95 (PSD-95) associate with components of the dynein motor complex and undergo retrograde cotransport with a dynein subunit. Transgenic depletion of dynein function in mice causes postsynaptic NLG1/3 and PSD-95 enrichment. In parallel, PSD lengths and spine head sizes are significantly increased, a phenotype similar to that observed upon transgenic overexpression of NLG1 (Dahlhaus et al., 2010). Moreover, application of a competitive PSD-95 peptide and neuroligin 1 C-terminal mutagenesis each specifically alter neuroligin 1 surface membrane expression and interfere with its internalization. Our data suggest the concept that synaptic plasticity regulates neuroligin turnover through active cytoskeleton transport.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Biotinilação , Células Cultivadas , Citoesqueleto/metabolismo , Proteína 4 Homóloga a Disks-Large , Dineínas/metabolismo , Eletrofisiologia , Guanilato Quinases , Hipocampo/citologia , Imuno-Histoquímica , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , TransfecçãoRESUMO
Different K(+) currents have been implicated in the myocardial action potential repolarization including the I(Kr). ERG1 alpha subunits, identified as the molecular correlate of I(Kr), have been shown to form heteromultimeric channels in the heart and their activity is modulated by a complex interplay of signal transduction events. Using electrophysiological techniques, we examined the effects of the cGMP-analogue 8-Br-cGMP on rat and guinea-pig papillary action potential duration (APD), on the biophysical properties of heterologously expressed homo- and heteromeric ERG1 channels, and on cardiac I(Kr). 8-Br-cGMP prolonged APD by about 25% after pharmacological inhibition of L-type Ca(2+) currents and I(Ks). The prolongation was completely abolished by prior application of the hERG channel blocker E-4031 or the protein kinase G (PKG) inhibitor Rp-8-Br-cGMPS. Expression analysis revealed the presence of both ERG1a and -1b subunits in rat papillary muscle. Both 8-Br-cGMP and ANP inhibited heterologously expressed ERG1b and even stronger ERG1a/1b channels, whereas ERG1a channels remained unaffected. The inhibitory 8-Br-cGMP effects were PKG-dependent and involved a profound ERG current reduction, which was also observed with cardiac AP clamp recordings. Measurements of I(Kr) from isolated mouse cardiomyocytes using Cs(+) as charge carrier exhibited faster deactivation kinetics in atrial than in ventricular myocytes consistent with a higher relative expression of ERG1b transcripts in atria than in ventricles. 8-Br-cGMP significantly reduced I(Kr) in atrial, but not in ventricular myocytes. These findings provide first evidence that through heteromeric assembly ERG1 channels become a critical target of cGMP-PKG signaling linking cGMP accumulation to cardiac I(Kr) modulation.
Assuntos
GMP Cíclico/metabolismo , Transdução de Sinais , Potenciais de Ação , Animais , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/farmacologia , Feminino , Cobaias , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Piperidinas , Piridinas , Ratos , Ratos Wistar , Tionucleotídeos , Transativadores , Regulador Transcricional ERGRESUMO
Flupirtine is an activator of Kv7 (KCNQ/M) potassium channels that has found clinical use as an analgesic with muscle relaxant properties. Kv7 potassium channels are expressed in axonal membranes and pharmacological activation of these channels may restore abnormal nerve excitability. We have examined the effect of flupirtine on the electrical excitability of myelinated axons in isolated segments of rat sural nerve. Axonal excitability was studied in vitro with the same parameters used by clinical neurophysiologists to assess peripheral nerve excitability in situ. Application of flupirtine in low micromolar concentrations resulted in an increase in threshold current, a reduction of refractoriness and an increase in post-spike superexcitability. These effects are consistent with an increase in Kv7 conductance and membrane hyperpolarization. Flupirtine also enhanced and prolonged the late, long-lasting period of axonal subexcitability that follows a short burst of action potentials. This effect was blocked by XE 991 (10 microM), an antagonist of Kv7 channels. In summary, flupirtine affects measures of excitability that are altered in the myelinated axons of patients with peripheral nerve disorders. This indicates that neuropathies with abnormal nerve excitability parameters corresponding to those affected by flupirtine may benefit from activation of axonal Kv7 potassium channels.
Assuntos
Aminopiridinas/farmacologia , Analgésicos/farmacologia , Axônios/efeitos dos fármacos , Canais de Potássio KCNQ/agonistas , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Nervo Sural/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Aminopiridinas/administração & dosagem , Analgésicos/administração & dosagem , Animais , Antracenos/farmacologia , Axônios/fisiologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Técnicas In Vitro , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microeletrodos , Fibras Nervosas Mielinizadas/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Nervo Sural/fisiologia , Fatores de TempoRESUMO
Different erg (ether-à-go-go-related gene; Kv11) K+ channel subunits are expressed throughout the brain. Especially mitral cells of the olfactory bulb are stained intensely by erg1a, erg1b, erg2, and erg3 antibodies. This led us to study the erg current in mitral/tufted (M/T) neurons from mouse olfactory bulb in primary culture. M/T neurons were identified by their morphology and presence of mGluR1 receptors, and RT-PCR demonstrated the expression of all erg subunits in cultured M/T neurons. Using an elevated external K+ concentration, a relatively uniform erg current was recorded in the majority of M/T cells and isolated with the erg channel blocker E-4031. With 4-s depolarizations, the erg current started to activate at -65 mV and exhibited half maximal activation at -51 mV. An increase in the external K+ concentration resulted in an increase in erg whole-cell conductance. The specific group 1 mGluR agonist, DHPG, which depolarizes mitral cells, reduced erg channel availability. DHPG accelerated erg current deactivation, reduced the maximum current amplitude, and shifted availability and activation curves to more depolarized potentials. A pharmacological block of erg channels depolarized the resting potential of M/T cells and clearly demonstrated the involvement of erg channels in the control of mitral cell excitability.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Neurônios/metabolismo , Bulbo Olfatório/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Antiarrítmicos/farmacologia , Células Cultivadas , Canal de Potássio ERG1 , Imuno-Histoquímica , Metoxi-Hidroxifenilglicol/análogos & derivados , Metoxi-Hidroxifenilglicol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Piridinas/farmacologia , Receptores de Glutamato Metabotrópico/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The actin cytoskeleton is crucial for function and morphology of neuronal synapses. Moreover, altered regulation of the neuronal actin cytoskeleton has been implicated in neuropsychiatric diseases such as autism spectrum disorder (ASD). Myosin XVI is a neuronally expressed unconventional myosin known to bind the WAVE regulatory complex (WRC), a regulator of filamentous actin (F-actin) polymerization. Notably, the gene encoding the myosin's heavy chain (MYO16) shows genetic association with neuropsychiatric disorders including ASD. Here, we investigated whether myosin XVI plays a role for actin cytoskeleton regulation in the dendritic spines of cerebellar Purkinje cells (PCs), a neuronal cell type crucial for motor learning, social cognition and vocalization. We provide evidence that both myosin XVI and the WRC component WAVE1 localize to PC spines. Fluorescence recovery after photobleaching (FRAP) analysis of GFP-actin in cultured PCs shows that Myo16 knockout as well as PC-specific Myo16 knockdown, lead to faster F-actin turnover in the dendritic spines of PCs. We also detect accelerated F-actin turnover upon interference with the WRC, and upon inhibition of Arp2/3 that drives formation of branched F-actin downstream of the WRC. In contrast, inhibition of formins that are responsible for polymerization of linear actin filaments does not cause faster F-actin turnover. Together, our data establish myosin XVI as a regulator of the postsynaptic actin cytoskeleton and suggest that it is an upstream activator of the WRC-Arp2/3 pathway in PC spines. Furthermore, ultra-structural and electrophysiological analyses of Myo16 knockout cerebellum reveals the presence of reduced numbers of synaptic vesicles at presynaptic terminals in the absence of the myosin. Therefore, we here define myosin XVI as an F-actin regulator important for presynaptic organization in the cerebellum.
RESUMO
TRAAK is a membrane tension-activated K+ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the 'leak' K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K+ channel.
Assuntos
Neurônios/enzimologia , Canais de Potássio/análise , Nós Neurofibrosos/enzimologia , Potenciais de Ação , Animais , Camundongos , Neurônios/fisiologia , RatosRESUMO
Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.
Assuntos
Cerebelo/metabolismo , Depressão Sináptica de Longo Prazo , Cadeias Pesadas de Miosina/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/fisiologia , Clatrina/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Endocitose/genética , Endocitose/fisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/genética , Células de Purkinje/metabolismo , Receptores de AMPA/agonistas , Receptores de AMPA/química , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
Gonadotropes are crucial in the control of reproduction but difficult to isolate for functional analysis due to their scattered distribution in the anterior pituitary gland. We devised a binary genetic approach, and describe a new mouse model that allows visualization and manipulation of gonadotrope cells. Using gene targeting in embryonic stem cells, we generated mice in which Cre recombinase is coexpressed with the GnRH receptor, which is expressed in gonadotrope cells. We show that we can direct Cre-mediated recombination of a yellow fluorescent protein reporter allele specifically in gonadotropes within the anterior pituitary of these knock-in mice. More than 99% of gonadotropin-containing cells were labeled by yellow fluorescent protein fluorescence and readily identifiable in dissociated pituitary cell culture, allowing potentially unbiased sampling from the gonadotrope population. Using electrophysiology, calcium imaging, and the study of secretion on the single-cell level, the functional properties of gonadotropes isolated from male mice were analyzed. Our studies demonstrate a significant heterogeneity in the resting properties of gonadotropes and their responses to GnRH. About 50% of gonadotropes do not exhibit secretion of LH or FSH. Application of GnRH induced a broad range of both electrophysiological responses and increases in the intracellular calcium concentration. Our mouse model will also be able to direct expression of other Cre recombination-dependent reporter genes to gonadotropes and, therefore, represents a versatile new tool in the understanding of gonadotrope biology.
Assuntos
Gonadotrofos/fisiologia , Gonadotropinas/genética , Receptores LHRH/genética , Animais , Cálcio/fisiologia , Eletrofisiologia , Éxons , Humanos , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Adeno-Hipófise/fisiologia , Potássio/fisiologiaRESUMO
TRPM4b is a Ca(2+)-activated, voltage-dependent monovalent cation channel that has been shown to act as a negative regulator of Ca(2+) entry and to be involved in the generation of oscillations of Ca(2+) influx in Jurkat T-lymphocytes. Transient overexpression of TRPM4b as an enhanced green fluorescence fusion protein in human embryonic kidney (HEK) cells resulted in its localization in the plasma membrane, as demonstrated by confocal fluorescence microscopy. The functionality and plasma membrane localization of overexpressed TRPM4b was confirmed by induction of Ca(2+)-dependent inward and outward currents in whole cell patch clamp recordings. HEK-293 cells stably overexpressing TRPM4b showed higher ionomycin-activated Ca(2+) influx than wild-type cells. In addition, analysis of the membrane potential using the potentiometric dye bis-(1,3-dibutylbarbituric acid)-trimethine oxonol and by current clamp experiments in the perforated patch configuration revealed a faster initial depolarization after activation of Ca(2+) entry with ionomycin. Furthermore, TRPM4b expression facilitated repolarization and thereby enhanced sustained Ca(2+) influx. In conclusion, in cells with a small negative membrane potential, such as HEK-293 cells, TRPM4b acts as a positive regulator of Ca(2+) entry.