RESUMEN
Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel's tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation.
Asunto(s)
Canales de Potasio Shaw , Sitios de Unión , Canales de Potasio Shaw/metabolismoRESUMEN
The recurrent variant KCNC1-p.Arg320His causes progressive myoclonus epilepsy (EPM) type 7, defined by progressive myoclonus, epilepsy, and ataxia, and is without effective treatment. KCNC1 encodes the voltage-gated potassium channel subunit Kv3.1, specifically expressed in high-frequency-firing neurons. Variant subunits act via loss of function; hence, EPM7 pathogenesis may involve impaired excitability of Kv3.1-expressing neurons, while enhancing Kv3 activity could represent a viable therapeutic strategy. We generate a mouse model, Kcnc1-p.Arg320His/+, which recapitulates the core features of EPM7, including progressive ataxia and seizure susceptibility. Kv3.1-expressing cerebellar granule cells and neocortical parvalbumin-positive GABAergic interneurons exhibit abnormalities consistent with Kv3 channel dysfunction. A Kv3-specific positive modulator (AUT00206) selectively enhances the firing frequency of Kv3.1-expressing neurons and improves motor function and seizure susceptibility in Kcnc1-Arg320His/+ mice. This work identifies a cellular and circuit basis of dysfunction in EPM7 and demonstrates that Kv3 positive modulators such as AUT00206 have therapeutic potential for the treatment of EPM7.
Asunto(s)
Epilepsias Mioclónicas Progresivas , Ratones , Animales , Epilepsias Mioclónicas Progresivas/genética , Ataxia/genética , Convulsiones/genética , Neuronas , EncéfaloRESUMEN
Various psychiatric diseases are characterized by aberrant cognition and emotional regulation. This includes inappropriately attributing affective salience to innocuous cues, which can be investigated using translationally relevant preclinical models of fear discrimination. Activity in the underpinning corticolimbic circuitry is governed by parvalbumin-expressing GABAergic interneurons, which also regulate fear discrimination. Kv3 voltage-gated potassium channels are highly expressed in these neurons and are important for controlling their activity, suggesting that pharmacological Kv3 modulation may regulate fear discrimination. We determined the effect of the positive Kv3 modulator AUT00206 given systemically to female rats undergoing limited or extended auditory fear discrimination training, which we have previously shown results in more discrimination or generalization, respectively, based on freezing at retrieval. We also characterized darting and other active fear-related responses. We found that limited training resulted in more discrimination based on freezing, which was unaffected by AUT00206. In contrast, extended training resulted in more generalization based on freezing and the emergence of discrimination based on darting during training and, to a lesser extent, at retrieval. Importantly, AUT00206 given before extended training had dissociable effects on fear discrimination and expression at retrieval depending on the response examined. While AUT00206 mitigated generalization without affecting expression based on freezing, it reduced expression without affecting discrimination based on darting, although darting levels were low overall. These results indicate that pharmacological Kv3 modulation regulates fear discrimination and expression in a response-dependent manner. They also raise the possibility that targeting Kv3 channels may ameliorate perturbed cognition and emotional regulation in psychiatric disease.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Ratas , Femenino , Animales , Canales de Potasio con Entrada de Voltaje/metabolismo , Neuronas/fisiología , Interneuronas/metabolismo , MiedoRESUMEN
Synapse loss is associated with cognitive decline in Alzheimer's disease, and owing to their plastic nature, synapses are an ideal target for therapeutic intervention. Oligomeric amyloid beta around amyloid plaques is known to contribute to synapse loss in mouse models and is associated with synapse loss in human Alzheimer's disease brain tissue, but the mechanisms leading from Aß to synapse loss remain unclear. Recent data suggest that the fast-activating and -inactivating voltage-gated potassium channel subtype 3.4 (Kv3.4) may play a role in Aß-mediated neurotoxicity. Here, we tested whether this channel could also be involved in Aß synaptotoxicity. Using adeno-associated virus and clustered regularly interspaced short palindromic repeats technology, we reduced Kv3.4 expression in neurons of the somatosensory cortex of APP/PS1 mice. These mice express human familial Alzheimer's disease-associated mutations in amyloid precursor protein and presenilin-1 and develop amyloid plaques and plaque-associated synapse loss similar to that observed in Alzheimer's disease brain. We observe that reducing Kv3.4 levels ameliorates dendritic spine loss and changes spine morphology compared to control virus. In support of translational relevance, Kv3.4 protein was observed in human Alzheimer's disease and control brain and is associated with synapses in human induced pluripotent stem cell-derived cortical neurons. We also noted morphological changes in induced pluripotent stem cell neurones challenged with human Alzheimer's disease-derived brain homogenate containing Aß but, in this in vitro model, total mRNA levels of Kv3.4 were found to be reduced, perhaps as an early compensatory mechanism for Aß-induced damage. Overall, our results suggest that approaches to reduce Kv3.4 expression and/or function in the Alzheimer's disease brain could be protective against Aß-induced synaptic alterations.
RESUMEN
Autonomic parasympathetic preganglionic neurons (PGNs) drive contraction of the bladder during micturition but remain quiescent during bladder filling. This quiescence is postulated to be because of recurrent inhibition of PGN by fast-firing adjoining interneurons. Here, we defined four distinct neuronal types within Lamina VII, where PGN are situated, by combining whole cell patch clamp recordings with k-means clustering of a range of electrophysiological parameters. Additional morphologic analysis separated these neuronal classes into parasympathetic preganglionic populations (PGN) and a fast-firing interneuronal population. Kv3 channels are voltage-gated potassium channels (Kv) that allow fast and precise firing of neurons. We found that blockade of Kv3 channels by tetraethylammonium (TEA) reduced neuronal firing frequency and isolated high-voltage-activated Kv currents in the fast-firing population but had no effect in PGN populations. Furthermore, Kv3 blockade potentiated the local and descending inhibitory inputs to PGN indicating that Kv3-expressing inhibitory neurons are synaptically connected to PGN. Taken together, our data reveal that Kv3 channels are crucial for fast and regulated neuronal output of a defined population that may be involved in intrinsic spinal bladder circuits that underpin recurrent inhibition of PGN.
Asunto(s)
Neuronas , Canales de Potasio Shaw , Potenciales de Acción/fisiología , Neuronas/fisiología , Técnicas de Placa-Clamp , Médula Espinal/fisiologíaRESUMEN
AUT00063 and AUT00202 are novel pharmaceutical modulators of the Kv3 subfamily of voltage-gated K+ channels. Kv3.1 channels, which control fast firing of many central auditory neurons, have been shown to decline with age and this may contribute to age-related deficits in central auditory processing. In the present study, the effects of the two novel compounds that specifically modulate Kv3 channels on auditory temporal processing were examined in aged (19-25-month-old) and young-adult (3-5 month-old) Fischer 344 rats (F344) using a behavioral gap-prepulse inhibition (gap-PPI) paradigm. The acoustic startle response (ASR) and its inhibition induced by a gap in noise were measured before and after drug administration. Hearing thresholds in tested rats were evaluated by the auditory brainstem response (ABR). Aged F344 rats had significantly higher ABR thresholds, lower amplitudes of ASR, and weaker gap-PPI compared with young-adult rats. No influence of AUT00063 and AUT00202 administration was observed on ABR hearing thresholds in rats of both age groups. AUT00063 and AUT00202 had suppressive effect on ASR of F344 rats that was more pronounced with AUT00063. The degree of suppression depended on the dose and age of the rats. Both compounds significantly improved the gap-PPI performance in gap detection tests in aged rats. These results indicate that AUT00063 and AUT00202 may influence intrinsic firing properties of neurons in the central auditory system of aged animals and have the potential to treat aged-related hearing disorders.
Asunto(s)
Percepción Auditiva , Potenciales Evocados Auditivos del Tronco Encefálico , Estimulación Acústica , Animales , Umbral Auditivo , Inhibición Prepulso , Ratas , Ratas Endogámicas F344 , Reflejo de Sobresalto , Canales de Potasio ShawRESUMEN
Tinnitus is unusual for such a common symptom in that there are few treatment options and those that are available are aimed at reducing the impact rather than specifically addressing the tinnitus percept. In particular, there is no drug recommended specifically for the management of tinnitus. Whilst some of the currently available interventions are effective at improving quality of life and reducing tinnitus-associated psychological distress, most show little if any effect on the primary symptom of subjective tinnitus loudness. Studies of the delivery of tinnitus services have demonstrated considerable end-user dissatisfaction and a marked disconnect between the aims of healthcare providers and those of tinnitus patients: patients want their tinnitus loudness reduced and would prefer a pharmacological solution over other modalities. Several studies have shown that tinnitus confers a significant financial burden on healthcare systems and an even greater economic impact on society as a whole. Market research has demonstrated a strong commercial opportunity for an effective pharmacological treatment for tinnitus, but the amount of tinnitus research and financial investment is small compared to other chronic health conditions. There is no single reason for this situation, but rather a series of impediments: tinnitus prevalence is unclear with published figures varying from 5.1 to 42.7%; there is a lack of a clear tinnitus definition and there are multiple subtypes of tinnitus, potentially requiring different treatments; there is a dearth of biomarkers and objective measures for tinnitus; treatment research is associated with a very large placebo effect; the pathophysiology of tinnitus is unclear; animal models are available but research in animals frequently fails to correlate with human studies; there is no clear definition of what constitutes meaningful change or "cure"; the pharmaceutical industry cannot see a clear pathway to distribute their products as many tinnitus clinicians are non-prescribing audiologists. To try and clarify this situation, highlight important areas for research and prevent wasteful duplication of effort, the British Tinnitus Association (BTA) has developed a Map of Tinnitus. This is a repository of evidence-based tinnitus knowledge, designed to be free to access, intuitive, easy to use, adaptable and expandable.
RESUMEN
AUT00063 is an experimental new medicine that has been demonstrated to suppress spontaneous hyperactivity by modulating the action of voltage-gated potassium-channels in central auditory cortical neurons of a rodent model. This neurobiological property makes it a good candidate for treating the central component of subjective tinnitus but this has not yet been tested in humans. The main purpose of the QUIET-1 (QUest In Eliminating Tinnitus) trial was to examine the effect of AUT00063 on the severity of tinnitus symptoms in people with subjective tinnitus. The trial was a randomised, placebo-controlled, observer, physician and participant blinded multi-centre superiority trial with two parallel groups and a primary endpoint of functional impact on tinnitus 28 days after the first drug dosing day. The trial design overcame the scale and logistical challenges of delivering a scientifically robust, statistically powered multi-centre study for subjective tinnitus within the National Health Service in England. The trial was terminated early for futility. Overall, 212 participants consented across 18 sites with 91 participants randomised to groups using age, gender, tinnitus symptom severity and hearing status as minimisation factors. While the pharmacokinetic markers confirm the uptake of AUT00063 in the body, within the expected therapeutic range, with respect to clinical benefit findings indicated that AUT00063 was not effective in alleviating tinnitus symptoms (1.56 point change in Tinnitus Functional Index). In terms of clinical harms, results indicated that a daily dose of 800â¯mg capsules of AUT00063 taken for 28 days was safe and well tolerated. These findings provide significant advances in the drug development field for hearing sciences, but raise questions about the predictive validity of certain rodent models of noise-induced hearing loss and tinnitus, as least for the mechanism evaluated in the present study. Trial Registration: (EudraCT) 2014-002179-27; NCT02315508.
Asunto(s)
Percepción Auditiva/efectos de los fármacos , Audición/efectos de los fármacos , Imidazoles/uso terapéutico , Pirimidinas/uso terapéutico , Canales de Potasio Shaw/efectos de los fármacos , Acúfeno/tratamiento farmacológico , Adulto , Anciano , Método Doble Ciego , Terminación Anticipada de los Ensayos Clínicos , Inglaterra , Femenino , Humanos , Imidazoles/efectos adversos , Imidazoles/farmacocinética , Masculino , Persona de Mediana Edad , Pirimidinas/efectos adversos , Pirimidinas/farmacocinética , Índice de Severidad de la Enfermedad , Canales de Potasio Shaw/metabolismo , Factores de Tiempo , Acúfeno/diagnóstico , Acúfeno/metabolismo , Acúfeno/fisiopatología , Resultado del TratamientoRESUMEN
Fragile X syndrome (FXS) is characterized by hypersensitivity to sensory stimuli, including environmental sounds. We compared the auditory brainstem response (ABR) recorded in vivo in mice lacking the gene (Fmr1-/y ) for fragile X mental retardation protein (FMRP) with that in wild-type animals. We found that ABR wave I, which represents input from the auditory nerve, is reduced in Fmr1-/y animals, but only at high sound levels. In contrast, wave IV, which represents the activity of auditory brainstem nuclei is enhanced at all sound levels, suggesting that loss of FMRP alters the central processing of auditory signals. Current-clamp recordings of neurons in the medial nucleus of the trapezoid body in the auditory brainstem revealed that, in contrast to neurons from wild-type animals, sustained depolarization triggers repetitive firing rather than a single action potential. In voltage-clamp recordings, K+ currents that activate at positive potentials ("high-threshold" K+ currents), which are required for high-frequency firing and are carried primarily by Kv3.1 channels, are elevated in Fmr1-/y mice, while K+ currents that activate near the resting potential and inhibit repetitive firing are reduced. We therefore tested the effects of AUT2 [((4-({5-[(4R)-4-ethyl-2,5-dioxo-1-imidazolidinyl]-2-pyridinyl}oxy)-2-(1-methylethyl) benzonitrile], a compound that modulates Kv3.1 channels. AUT2 reduced the high-threshold K+ current and increased the low-threshold K+ currents in neurons from Fmr1-/y animals by shifting the activation of the high-threshold current to more negative potentials. This reduced the firing rate and, in vivo, restored wave IV of the ABR. Our results from animals of both sexes suggest that the modulation of the Kv3.1 channel may have potential for the treatment of sensory hypersensitivity in patients with FXS.SIGNIFICANCE STATEMENT mRNA encoding the Kv3.1 potassium channel was one of the first described targets of the fragile X mental retardation protein (FMRP). Fragile X syndrome is caused by loss of FMRP and, in humans and mice, causes hypersensitivity to auditory stimuli. We found that components of the auditory brain response (ABR) corresponding to auditory brainstem activity are enhanced in mice lacking FMRP. This is accompanied by hyperexcitability and altered potassium currents in auditory brainstem neurons. Treatment with a drug that alters the voltage dependence of Kv3.1 channels normalizes the imbalance of potassium currents, as well as ABR responses in vivo, suggesting that such compounds may be effective in treating some symptoms of fragile X syndrome.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Canales de Potasio Shaw/metabolismo , Animales , Vías Auditivas , Percepción Auditiva , Tronco Encefálico/efectos de los fármacos , Núcleo Coclear/fisiología , Fenómenos Electrofisiológicos , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Femenino , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/genética , Hidantoínas/farmacología , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Piridinas/farmacologíaRESUMEN
Temporal processing by cochlear implant listeners is degraded and is affected by auditory deprivation. The fast-acting Kv3.1 potassium channel is important for sustained temporally accurate firing and is also susceptible to deprivation, the effects of which can be partially restored in animals by the molecule AUT00063. We report the results of a randomised placebo-controlled double-blind study on psychophysical tests of the effects of AUT00063 on temporal processing by CI listeners. The study measured the upper limit of temporal pitch, gap detection, and discrimination of low rates (centred on 120 pps) for monopolar pulse trains presented to an apical electrode. The upper limit was measured using the optimally efficient midpoint comparison (MPC) pitch-ranking procedure; thresholds were obtained for the other two measures using an adaptive procedure. Twelve CI users (MedEl and Cochlear) were tested before and after two periods of AUT00063 or placebo in a within-subject crossover study. No significant differences occurred between post-drug and post-placebo conditions. This absence of effect occurred despite high test-retest reliability for all three measures, obtained by comparing performance on the two baseline visits, and despite the demonstrated sensitivity of the measures to modest changes in temporal processing obtained in other studies from our laboratory. Hence, we have no evidence that AUT00063 improves temporal processing for the doses and patient population employed.
Asunto(s)
Percepción Auditiva/efectos de los fármacos , Implantes Cocleares , Sordera/terapia , Imidazoles/uso terapéutico , Pirimidinas/uso terapéutico , Anciano , Anciano de 80 o más Años , Estudios Cruzados , Método Doble Ciego , Pruebas Auditivas , Humanos , Imidazoles/farmacología , Persona de Mediana Edad , Pirimidinas/farmacología , Canales de Potasio ShawRESUMEN
Noise exposure has been shown to produce long-lasting increases in spontaneous activity in central auditory structures in animal models, and similar pathologies are thought to contribute to clinical phenomena such as hyperacusis or tinnitus in humans. Here we demonstrate that multi-unit spontaneous neuronal activity in the inferior colliculus (IC) of mice is significantly elevated four weeks following noise exposure at recording sites with frequency tuning within or near the noise exposure band, and this selective central auditory pathology can be normalised through administration of a novel compound that modulates activity of Kv3 voltage-gated ion channels. The compound had no statistically significant effect on IC spontaneous activity without noise exposure, nor on thresholds or frequency tuning of tone-evoked responses either with or without noise exposure. Administration of the compound produced some reduction in the magnitude of evoked responses to a broadband noise, but unlike effects on spontaneous rates, these effects on evoked responses were not specific to recording sites with frequency tuning within the noise exposure band. Thus, the results suggest that modulators of Kv3 channels can selectively counteract increases in spontaneous activity in the auditory midbrain associated with noise exposure.
Asunto(s)
Estimulación Acústica/métodos , Potenciales Evocados Auditivos del Tronco Encefálico/efectos de los fármacos , Imidazoles/farmacología , Colículos Inferiores/efectos de los fármacos , Pirimidinas/farmacología , Canales de Potasio Shaw/efectos de los fármacos , Animales , Vías Auditivas/efectos de los fármacos , Vías Auditivas/metabolismo , Umbral Auditivo/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/farmacocinética , Colículos Inferiores/metabolismo , Masculino , Ratones Endogámicos CBA , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Pirimidinas/farmacocinética , Canales de Potasio Shaw/genética , Canales de Potasio Shaw/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Exposure to loud sound increases burst-firing of dorsal cochlear nucleus (DCN) fusiform cells in the auditory brainstem, which has been suggested to be an electrophysiological correlate of tinnitus. The altered activity of DCN fusiform cells may be due to down-regulation of high voltage-activated (Kv3-like) K+ currents. Whole cell current-clamp recordings were obtained from DCN fusiform cells in brain slices from P15-P18 CBA mice. We first studied whether acoustic over-exposure (performed at P15) or pharmacological inhibition of K+ currents with tetraethylamonium (TEA) affect fusiform cell action potential characteristics, firing frequency and spike-timing relative to evoking current stimuli. We then tested whether AUT1, a modulator of Kv3 K+ currents reverses the effects of sound exposure or TEA. Both loud sound exposure and TEA decreased the amplitude of action potential after-hyperpolarization, reduced the maximum firing frequency, and disrupted spike-timing. These treatments also increased post-synaptic voltage fluctuations at baseline. AUT1 applied in the presence of TEA or following acoustic over-exposure, did not affect the firing frequency, but enhanced action potential after-hyperpolarization, prevented the increased voltage fluctuations and restored spike-timing. Furthermore AUT1 prevented the occurrence of bursts. Our study shows that the effect on spike-timing is significantly correlated with the amplitude of the action potential after-hyperpolarization and the voltage fluctuations at baseline. In conclusion, modulation of putative Kv3 K+ currents may restore regular spike-timing of DCN fusiform cell firing following noise exposure, and could provide a means to restore deficits in temporal encoding observed during noise-induced tinnitus.
Asunto(s)
Potenciales de Acción/fisiología , Núcleo Coclear/citología , Neuronas/fisiología , Canales de Potasio Shaw/metabolismo , Estimulación Acústica , Potenciales de Acción/efectos de los fármacos , Análisis de Varianza , Animales , Animales Recién Nacidos , Interacciones Farmacológicas , Femenino , Hidantoínas/farmacología , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos CBA , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Inhibición Prepulso/efectos de los fármacos , Inhibición Prepulso/fisiología , Piridinas/farmacología , Tetraetilamonio/farmacología , Factores de TiempoRESUMEN
Kv3.1 and Kv3.2 voltage-gated potassium channels are expressed on parvalbumin-positive GABAergic interneurons in corticolimbic brain regions and contribute to high-frequency neural firing. The channels are also expressed on GABAergic neurons of the basal ganglia, substantia nigra, and ventral tegmental area (VTA) where they regulate firing patterns critical for movement control, reward, and motivation. Modulation of Kv3.1 and Kv3.2 channels may therefore have potential in the treatment of disorders in which these systems have been implicated, such as bipolar disorder. Following the recent development of a potassium channel modulator, AUT1-an imidazolidinedione compound that specifically increases currents mediated by Kv3.1 and Kv3.2 channels in recombinant systems-we report that the compound is able to reverse 'manic-like' behavior in two mouse models: amphetamine-induced hyperactivity and ClockΔ19 mutants. AUT1 completely prevented amphetamine-induced hyperactivity in a dose-dependent manner, similar to the atypical antipsychotic, clozapine. Similar efficacy was observed in Kv3.2 knockout mice. In contrast, AUT1 was unable to prevent amphetamine-induced hyperactivity in mice lacking Kv3.1 channels. Notably, Kv3.1-null mice displayed baseline hyperlocomotion, reduced anxiety-like behavior, and antidepressant-like behavior. In ClockΔ19 mice, AUT1 reversed hyperactivity. Furthermore, AUT1 application modulated firing frequency and action potential properties of ClockΔ19 VTA dopamine neurons potentially through network effects. Kv3.1 protein levels in the VTA of ClockΔ19 and WT mice were unaltered by acute AUT1 treatment. Taken together, these results suggest that the modulation of Kv3.1 channels may provide a novel approach to the treatment of bipolar mania.
Asunto(s)
Acatisia Inducida por Medicamentos/tratamiento farmacológico , Conducta Animal/efectos de los fármacos , Trastorno Bipolar/tratamiento farmacológico , Proteínas CLOCK , Neuronas Dopaminérgicas/efectos de los fármacos , Hidantoínas/farmacología , Piridinas/farmacología , Canales de Potasio Shaw/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismo , Anfetamina/farmacología , Animales , Proteínas CLOCK/genética , Estimulantes del Sistema Nervioso Central/farmacología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Canales de Potasio Shaw/deficienciaRESUMEN
Higher stages of central auditory processing compensate for a loss of cochlear nerve synapses by increasing the gain on remaining afferent inputs, thereby restoring firing rate codes for rudimentary sound features. The benefits of this compensatory plasticity are limited, as the recovery of precise temporal coding is comparatively modest. We reasoned that persistent temporal coding deficits could be ameliorated through modulation of voltage-gated potassium (Kv) channels that regulate temporal firing patterns. Here, we characterize AUT00063, a pharmacological compound that modulates Kv3.1, a high-threshold channel expressed in fast-spiking neurons throughout the central auditory pathway. Patch clamp recordings from auditory brainstem neurons and in silico modeling revealed that application of AUT00063 reduced action potential timing variability and improved temporal coding precision. Systemic injections of AUT00063 in vivo improved auditory synchronization and supported more accurate decoding of temporal sound features in the inferior colliculus and auditory cortex in adult mice with a near-complete loss of auditory nerve afferent synapses in the contralateral ear. These findings suggest modulating Kv3.1 in central neurons could be a promising therapeutic approach to mitigate temporal processing deficits that commonly accompany aging, tinnitus, ototoxic drug exposure or noise damage.
Asunto(s)
Percepción Auditiva/efectos de los fármacos , Imidazoles/farmacología , Moduladores del Transporte de Membrana/farmacología , Mesencéfalo/efectos de los fármacos , Pirimidinas/farmacología , Canales de Potasio Shaw/metabolismo , Enfermedades del Nervio Vestibulococlear/tratamiento farmacológico , Potenciales de Acción/efectos de los fármacos , Animales , Vías Auditivas/efectos de los fármacos , Vías Auditivas/lesiones , Vías Auditivas/metabolismo , Percepción Auditiva/fisiología , Nervio Coclear/lesiones , Nervio Coclear/metabolismo , Conducta Compulsiva , Modelos Animales de Enfermedad , Mesencéfalo/metabolismo , Ratones , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ouabaína , Recuperación de la Función/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Enfermedades del Nervio Vestibulococlear/metabolismoRESUMEN
Many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem, express "high threshold" voltage-gated Kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate Kv3.1 channels. Using Chinese hamster ovary cells stably expressing rat Kv3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of Kv3.1 currents toward negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single-channel recordings also showed that AUT1 shifted the open probability of Kv3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of Kv3.1 currents represents a novel avenue for manipulation of neuronal excitability and has the potential for therapeutic benefit in the treatment of hearing disorders.
Asunto(s)
Tronco Encefálico/efectos de los fármacos , Hidantoínas/farmacología , Neuronas/efectos de los fármacos , Neurotransmisores/farmacología , Piridinas/farmacología , Canales de Potasio Shaw/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Tronco Encefálico/fisiología , Células CHO , Simulación por Computador , Cricetulus , Hidantoínas/química , Ratones Endogámicos C57BL , Modelos Moleculares , Modelos Neurológicos , Estructura Molecular , Neuronas/fisiología , Neurotransmisores/química , Técnicas de Placa-Clamp , Piridinas/química , Ratas , Canales de Potasio Shaw/genética , Técnicas de Cultivo de TejidosRESUMEN
Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Canales de Potasio Shaw/metabolismo , Ataxias Espinocerebelosas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Células Madre Pluripotentes/metabolismo , Canales de Potasio Shaw/química , Canales de Potasio Shaw/genética , Transducción de Señal , Proteínas de Unión al GTP rac/metabolismoRESUMEN
Kv3.1 and Kv3.2 high voltage-activated potassium channels, which display fast activation and deactivation kinetics, are known to make a crucial contribution to the fast-spiking phenotype of certain neurons. Pharmacological experiments show that the blockade of native Kv3 currents with low concentrations of tetraethylammonium or 4-aminopyridine impairs the expression of this firing phenotype. In particular, Kv3 channels are highly expressed by fast-spiking, parvalbumin-positive interneurons in corticolimbic brain circuits, which modulate the synchronization of cortical circuits and the generation of brain rhythms. Here, we describe a novel small molecule, (5R)-5-ethyl-3-(6-{[4-methyl-3-(methyloxy)phenyl]oxy}-3-pyridinyl)-2,4-imidazolidinedione (AUT1), which modulates Kv3.1 and Kv3.2 channels in human recombinant and rodent native neurons. AUT1 increased whole currents mediated by human Kv3.1b and Kv3.2a channels, with a concomitant leftward shift in the voltage dependence of activation. A less potent effect was observed on hKv3.3 currents. In mouse somatosensory cortex slices in vitro, AUT1 rescued the fast-spiking phenotype of parvalbumin-positive-fast-spiking interneurons following an impairment of their firing capacity by blocking a proportion of Kv3 channels with a low concentration of tetraethylammonium. Notably, AUT1 had no effect on interneuron firing when applied alone. Together, these data confirm the role played by Kv3 channels in the regulation of the firing phenotype of somatosensory interneurons and suggest that AUT1 and other Kv3 modulators could represent a new and promising therapeutic approach to the treatment of disorders associated with dysfunction of inhibitory feedback in corticolimbic circuits, such as schizophrenia.
Asunto(s)
Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Parvalbúminas/metabolismo , Canales de Potasio Shaw/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Células CHO , Línea Celular , Cricetulus , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes/metabolismo , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/metabolismo , Tetraetilamonio/farmacologíaRESUMEN
Exposure to loud sound causes cochlear damage resulting in hearing loss and tinnitus. Tinnitus has been related to hyperactivity in the central auditory pathway occurring weeks after loud sound exposure. However, central excitability changes concomitant to hearing loss and preceding those periods of hyperactivity, remain poorly explored. Here we investigate mechanisms contributing to excitability changes in the dorsal cochlear nucleus (DCN) shortly after exposure to loud sound that produces hearing loss. We show that acoustic overexposure alters synaptic transmission originating from the auditory and the multisensory pathway within the DCN in different ways. A reduction in the number of myelinated auditory nerve fibers leads to a reduced maximal firing rate of DCN principal cells, which cannot be restored by increasing auditory nerve fiber recruitment. In contrast, a decreased membrane resistance of DCN granule cells (multisensory inputs) leads to a reduced maximal firing rate of DCN principal cells that is overcome when additional multisensory fibers are recruited. Furthermore, gain modulation by inhibitory synaptic transmission is disabled in both auditory and multisensory pathways. These cellular mechanisms that contribute to decreased cellular excitability in the central auditory pathway are likely to represent early neurobiological markers of hearing loss and may suggest interventions to delay or stop the development of hyperactivity that has been associated with tinnitus.
Asunto(s)
Nervio Coclear/fisiopatología , Núcleo Coclear/fisiopatología , Pérdida Auditiva Provocada por Ruido/fisiopatología , Acúfeno/fisiopatología , Animales , Vías Auditivas/patología , Vías Auditivas/fisiopatología , Umbral Auditivo/fisiología , Cóclea/patología , Cóclea/fisiopatología , Nervio Coclear/patología , Núcleo Coclear/patología , Potenciales Postsinápticos Excitadores/fisiología , Pérdida Auditiva Provocada por Ruido/patología , Humanos , Potenciales Postsinápticos Inhibidores/fisiología , Fibras Nerviosas Mielínicas/patología , Fibras Nerviosas Mielínicas/fisiología , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Acúfeno/patologíaRESUMEN
Retigabine [RTG (international nonproprietary name); ezogabine (EZG; U.S. adopted name)] is a first-in-class antiepileptic drug (AED) that reduces neuronal excitability by enhancing the activity of KCNQ (K(v)7) potassium (K(+)) channels. RTG/EZG has recently been approved by the European Medicines Agency and the U.S. Food and Drug Administration as adjunctive therapy in adults with partial-onset seizures. In this review we discuss the activity that RTG/EZG has demonstrated across a broad spectrum of in vitro/in vivo animal models of seizures, including generalized tonic-clonic, primary generalized (absence), and partial seizures, in addition to the compound's ability to resist and block the occurrence of seizures induced by a range of stimuli across different regions of the brain. The potency of RTG/EZG in models refractory to several conventional AEDs and the work done to assess antiepileptogenesis and neuroprotection are discussed. Studies that have evaluated the central nervous system side effects of RTG/EZG in animals are reviewed in order to compare these effects with adverse events observed in patients with epilepsy. Based on its demonstrated effect in a number of animal epilepsy models, the synergistic and additive activity of RTG/EZG with other AEDs supports its potential use in therapeutic combinations for different seizure types. The distinct mechanism of action of RTG/EZG from those of currently available AEDs, along with its broad preclinical activity, underscores the key role of KCNQ (K(v)7) K(+) channels in neuronal excitability, and further supports the potential efficacy of this unique molecule in the treatment of epilepsy.
Asunto(s)
Anticonvulsivantes/farmacología , Carbamatos/farmacología , Modelos Animales de Enfermedad , Epilepsia/tratamiento farmacológico , Fenilendiaminas/farmacología , Animales , Anticonvulsivantes/efectos adversos , Anticonvulsivantes/uso terapéutico , Carbamatos/efectos adversos , Carbamatos/uso terapéutico , Sinergismo Farmacológico , Epilepsia/clasificación , Humanos , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fenilendiaminas/efectos adversos , Fenilendiaminas/uso terapéutico , Resultado del TratamientoRESUMEN
The pharmacologic profile of retigabine [RTG (international nonproprietary name); ezogabine, EZG (U.S. adopted name)], is different from all currently approved antiepileptic drugs (AEDs). Its primary mechanism of action (MoA) as a positive allosteric modulator of KCNQ2-5 (K(v) 7.2-7.5) ion channels defines RTG/EZG as the first neuronal potassium (K(+)) channel opener for the treatment of epilepsy. KCNQ2-5 channels are predominantly expressed in neurons and are important determinants of cellular excitability, as indicated by the occurrence of human genetic mutations in KCNQ channels that underlie inheritable disorders including, in the case of KCNQ2/3, the syndrome of benign familial neonatal convulsions. In vitro pharmacologic studies demonstrate that the most potent action of RTG/EZG is at KCNQ2-5 channels, particularly heteromeric KCNQ2/3. Furthermore, mutagenesis and modeling studies have pinpointed the RTG/EZG binding site to a hydrophobic pocket near the channel gate, indicating how RTG/EZG can stabilize the open form of KCNQ2-5 channels; the absence of this site in KCNQ1 also provides a clear explanation for the inbuilt selectivity RTG/EZG has for potassium channels other than the KCNQ cardiac channel. KCNQ channels are active at the normal cell resting membrane potential (RMP) and contribute a continual hyperpolarizing influence that stabilizes cellular excitability. The MoA of RTG/EZG increases the number of KCNQ channels that are open at rest and also primes the cell to retort with a larger, more rapid, and more prolonged response to membrane depolarization or increased neuronal excitability. In this way, RTG/EZG amplifies this natural inhibitory force in the brain, acting like a brake to prevent the high levels of neuronal action potential burst firing (epileptiform activity) that may accompany sustained depolarizations associated with the initiation and propagation of seizures. This action to restore physiologic levels of neuronal activity is thought to underlie the efficacy of RTG/EZG as an anticonvulsant in a broad spectrum of preclinical seizure models and in placebo-controlled trials in patients with partial epilepsy. In this article, we consider the pharmacologic characteristics of RTG/EZG at the receptor, cellular, and network levels as a means of understanding the novel and efficacious MoA of this new AED as defined in both preclinical and clinical research.