RESUMEN
BACKGROUND: Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. METHODS: We generated mice deleted for Cav 1.2 in T cells or Cav 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. RESULTS: We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3 must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. CONCLUSIONS: Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.
Asunto(s)
Asma , Canales de Calcio , Animales , Asma/metabolismo , Calcio/metabolismo , Canales de Calcio/metabolismo , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Ratones , Receptores de Antígenos de Linfocitos T/metabolismo , Células Th2/metabolismoRESUMEN
Dysfunction of pacemaker activity in the sinoatrial node (SAN) underlies "sick sinus" syndrome (SSS), a common clinical condition characterized by abnormally low heart rate (bradycardia). If untreated, SSS carries potentially life-threatening symptoms, such as syncope and end-stage organ hypoperfusion. The only currently available therapy for SSS consists of electronic pacemaker implantation. Mice lacking L-type Cav1.3 Ca(2+) channels (Cav1.3(-/-)) recapitulate several symptoms of SSS in humans, including bradycardia and atrioventricular (AV) dysfunction (heart block). Here, we tested whether genetic ablation or pharmacological inhibition of the muscarinic-gated K(+) channel (IKACh) could rescue SSS and heart block in Cav1.3(-/-) mice. We found that genetic inactivation of IKACh abolished SSS symptoms in Cav1.3(-/-) mice without reducing the relative degree of heart rate regulation. Rescuing of SAN and AV dysfunction could be obtained also by pharmacological inhibition of IKACh either in Cav1.3(-/-) mice or following selective inhibition of Cav1.3-mediated L-type Ca(2+) (ICa,L) current in vivo. Ablation of IKACh prevented dysfunction of SAN pacemaker activity by allowing net inward current to flow during the diastolic depolarization phase under cholinergic activation. Our data suggest that patients affected by SSS and heart block may benefit from IKACh suppression achieved by gene therapy or selective pharmacological inhibition.
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Canales de Calcio Tipo L/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Bloqueo Cardíaco/tratamiento farmacológico , Activación del Canal Iónico/fisiología , Síndrome del Seno Enfermo/tratamiento farmacológico , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Humanos , Ratones , Ratones NoqueadosRESUMEN
Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type Ca(V)1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (Ca(V)3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (Ca(V)2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., Ca(V)1.2 and Ca(V)1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective Ca(V)1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson's disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/farmacología , Canales de Calcio/fisiología , Canales de Calcio/clasificación , Canales de Calcio/genética , Canales de Calcio Tipo L/farmacología , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo N/farmacología , Canales de Calcio Tipo N/fisiología , Canales de Calcio Tipo T/farmacología , Canales de Calcio Tipo T/fisiología , Enfermedades Cardiovasculares/fisiopatología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Trastornos de la Audición/fisiopatología , Humanos , Enfermedades Metabólicas/fisiopatología , Enfermedades del Sistema Nervioso/fisiopatología , Ceguera Nocturna/fisiopatología , Fosfolípidos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
We have generated a novel, neuro-specific ncRNA microarray, covering 1472 ncRNA species, to investigate their expression in different mouse models for central nervous system diseases. Thereby, we analyzed ncRNA expression in two mouse models with impaired calcium channel activity, implicated in Epilepsy or Parkinson's disease, respectively, as well as in a mouse model mimicking pathophysiological aspects of Alzheimer's disease. We identified well over a hundred differentially expressed ncRNAs, either from known classes of ncRNAs, such as miRNAs or snoRNAs or which represented entirely novel ncRNA species. Several differentially expressed ncRNAs in the calcium channel mouse models were assigned as miRNAs and target genes involved in calcium signaling, thus suggesting feedback regulation of miRNAs by calcium signaling. In the Alzheimer mouse model, we identified two snoRNAs, whose expression was deregulated prior to amyloid plaque formation. Interestingly, the presence of snoRNAs could be detected in cerebral spine fluid samples in humans, thus potentially serving as early diagnostic markers for Alzheimer's disease. In addition to known ncRNAs species, we also identified 63 differentially expressed, entirely novel ncRNA candidates, located in intronic or intergenic regions of the mouse genome, genomic locations, which previously have been shown to harbor the majority of functional ncRNAs.
Asunto(s)
Enfermedad de Alzheimer/genética , Epilepsia/genética , MicroARNs/biosíntesis , Enfermedad de Parkinson/genética , ARN no Traducido/biosíntesis , Enfermedad de Alzheimer/patología , Animales , Canales de Calcio/genética , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Epilepsia/patología , Regulación de la Expresión Génica , Genoma , Humanos , Ratones , MicroARNs/genética , Especificidad de Órganos , Enfermedad de Parkinson/patología , ARN no Traducido/genética , Análisis de Matrices TisularesRESUMEN
Dopamine midbrain neurons within the substantia nigra are particularly prone to degeneration in Parkinson's disease. Their selective loss causes the major motor symptoms of Parkinson's disease, but the causes for the high vulnerability of SN DA neurons, compared to neighbouring, more resistant ventral tegmental area dopamine neurons, are still unclear. Consequently, there is still no cure available for Parkinson's disease. Current therapies compensate the progressive loss of dopamine by administering its precursor l-DOPA and/or dopamine D2-receptor agonists. D2-autoreceptors and Cav1.3-containing L-type Ca(2+) channels both contribute to Parkinson's disease pathology. L-type Ca(2+) channel blockers protect SN DA neurons from degeneration in Parkinson's disease and its mouse models, and they are in clinical trials for neuroprotective Parkinson's disease therapy. However, their physiological functions in SN DA neurons remain unclear. D2-autoreceptors tune firing rates and dopamine release of SN DA neurons in a negative feedback loop through activation of G-protein coupled potassium channels (GIRK2, or KCNJ6). Mature SN DA neurons display prominent, non-desensitizing somatodendritic D2-autoreceptor responses that show pronounced desensitization in PARK-gene Parkinson's disease mouse models. We analysed surviving human SN DA neurons from patients with Parkinson's disease and from controls, and detected elevated messenger RNA levels of D2-autoreceptors and GIRK2 in Parkinson's disease. By electrophysiological analysis of postnatal juvenile and adult mouse SN DA neurons in in vitro brain-slices, we observed that D2-autoreceptor desensitization is reduced with postnatal maturation. Furthermore, a transient high-dopamine state in vivo, caused by one injection of either l-DOPA or cocaine, induced adult-like, non-desensitizing D2-autoreceptor responses, selectively in juvenile SN DA neurons, but not ventral tegmental area dopamine neurons. With pharmacological and genetic tools, we identified that the expression of this sensitized D2-autoreceptor phenotype required Cav1.3 L-type Ca(2+) channel activity, internal Ca(2+), and the interaction of the neuronal calcium sensor NCS-1 with D2-autoreceptors. Thus, we identified a first physiological function of Cav1.3 L-type Ca(2+) channels in SN DA neurons for homeostatic modulation of their D2-autoreceptor responses. L-type Ca(2+) channel activity however, was not important for pacemaker activity of mouse SN DA neurons. Furthermore, we detected elevated substantia nigra dopamine messenger RNA levels of NCS-1 (but not Cav1.2 or Cav1.3) after cocaine in mice, as well as in remaining human SN DA neurons in Parkinson's disease. Thus, our findings provide a novel homeostatic functional link in SN DA neurons between Cav1.3- L-type-Ca(2+) channels and D2-autoreceptor activity, controlled by NCS-1, and indicate that this adaptive signalling network (Cav1.3/NCS-1/D2/GIRK2) is also active in human SN DA neurons, and contributes to Parkinson's disease pathology. As it is accessible to pharmacological modulation, it provides a novel promising target for tuning substantia nigra dopamine neuron activity, and their vulnerability to degeneration.
Asunto(s)
Autorreceptores/metabolismo , Canales de Calcio Tipo L/fisiología , Neuronas Dopaminérgicas/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Proteínas Sensoras del Calcio Neuronal/fisiología , Neuropéptidos/fisiología , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Área Tegmental Ventral/metabolismo , Animales , Señalización del Calcio/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Receptores de Dopamina D2/metabolismo , Sustancia Negra/citología , Sustancia Negra/patología , Área Tegmental Ventral/citología , Área Tegmental Ventral/patologíaRESUMEN
Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Ca(v)1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (V(rest)), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Ca(v)1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Ca(v)1.3(-/-) mice, we investigated the role of Ca(v)1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1-3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Ca(v)1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Ca(v)1.3 to the subthreshold Ca(2+) charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Ca(v)1.3(-/-) MCCs. Low adaptation ratios due to reduced SK activation associated with Ca(v)1.3 deficiency prevent the efficient recovery of Na(V) channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Ca(v)1.3 slows down MCC firing by activating SK channels that maintain Na(V) channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.
Asunto(s)
Adaptación Fisiológica/fisiología , Canales de Calcio Tipo L/fisiología , Células Cromafines/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Potenciales de Acción/fisiología , Adaptación Fisiológica/efectos de los fármacos , Animales , Apamina/farmacología , Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Canales de Calcio Tipo L/efectos de los fármacos , Células Cromafines/efectos de los fármacos , ADN Complementario/biosíntesis , ADN Complementario/genética , Fenómenos Electrofisiológicos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Placa-Clamp , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Sodio/efectos de los fármacosRESUMEN
AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesoaccumbal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization, we have examined phosphorylation of GluA1 at key residues serine 845 (S845) and S831, as well as GluA1 cell surface levels in the nucleus accumbens (NAc) of cocaine-preexposed mice and the role of brain-specific Ca(v)1.2 and Ca(v)1.3 L-type Ca²âº channels (LTCCs), therein. We found higher basal levels of S845 phospho-GluA1 (P-GluA1) and cell surface GluA1 in the NAc following protracted withdrawal from cocaine exposure, changes that occur independently of LTCCs. In contrast, we found that a cocaine challenge that elicits expression of the cocaine-sensitized response increases S831 P-GluA1 that further increases surface GluA1 beyond the higher basal levels. Intra-NAc pharmacological manipulations indicate that the Ca(v)1.2-activated CaM kinase II (CaMKII) mediates cocaine-induced increase in S831 P-GluA1 and that both Ca(v)1.2-activated CaMKII and extracellular signal-regulated kinase 2 (ERK2) mediate the increase in GluA1 cell surface levels specific to the sensitized response. Experiments using adenoassociated viral vectors expressing Ca(v)1.3 and ERK2 siRNA further indicate that recruitment of the Ca(v)1.2 pathway in the NAc is dependent on ventral tegmental area Ca(v)1.3 LTCCs and ERK2. Together, these results identify candidate pathways that mediate cocaine-induced AMPAR plasticity in the NAc and provide a mechanism linking LTCCs and GluA1 plasticity to cocaine-induced persistent behavioral changes.
Asunto(s)
Adaptación Fisiológica/fisiología , Canales de Calcio Tipo L/fisiología , Cocaína/farmacología , Núcleo Accumbens/fisiología , Receptores AMPA/metabolismo , Área Tegmental Ventral/fisiología , Adaptación Fisiológica/efectos de los fármacos , Animales , Canales de Calcio Tipo L/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Dependovirus/genética , Vectores Genéticos , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , Receptores AMPA/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
Dysregulation of L-type Ca(2+) currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca(v)1.2 and Ca(v)1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca(v)1.2 and Ca(v)1.3 channels in mouse SAN cells and determined how these properties of Ca(v)1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba(2+) currents were recorded from SAN cells from mice carrying a point mutation that renders Ca(v)1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca(v)1.2-mediated current was isolated in the presence of nimodipine (1 µm), which was subtracted from the total current to yield the Ca(v)1.3 component. With strong depolarizations (+80 mV), Ca(v)1.2 underwent significantly stronger inactivation than Ca(v)1.3. VDF of Ca(v)1.3 was evident during recovery from inactivation at a time when Ca(v)1.2 remained inactivated. By immunofluorescence, Ca(v)1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca(v)1.2 was largely restricted to the delimiting plasma membrane. Ca(v)1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca(v)1.3 to ryanodine receptor-mediated Ca(2+) release. We conclude that strong VDF and colocalization with ryanodine receptors in mouse SAN cells are unique properties that may underlie a specific role for Ca(v)1.3 in opposing abnormal slowing of heart rate.
Asunto(s)
Relojes Biológicos , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Frecuencia Cardíaca , Nodo Sinoatrial/metabolismo , Animales , Relojes Biológicos/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/genética , Señalización del Calcio/efectos de los fármacos , Simulación por Computador , Dihidropiridinas/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Potenciales de la Membrana , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Cardiovasculares , Técnicas de Placa-Clamp , Mutación Puntual , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Sarcómeros/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Factores de TiempoRESUMEN
L-type Ca(2+) channel (LTCC)-activated signaling cascades contribute significantly to psychostimulant-induced locomotor sensitization; however, the precise contribution of the two brain-specific subunits Ca(v)1.2 and Ca(v)1.3 remains mostly unknown. In this study, by using amphetamine and cocaine locomotor sensitization in mutant mice expressing dihydropyridine (DHP)-insensitive Ca(v)1.2 LTCCs (Ca(v)1.2DHP(-/-)), we find that, as opposed to a previously identified role of the Ca(v)1.3 subunit of LTCCs in development of sensitization, the Ca(v)1.2 subunit mediates expression of amphetamine and cocaine sensitization when examined after a 14 d drug-free period. Molecular studies to further elucidate the role of Ca(v)1.2 versus Ca(v)1.3 LTCCs in activating signaling pathways in the nucleus accumbens (NAc) of drug-naive versus drug-preexposed mice examined 14 d later revealed that an acute amphetamine and cocaine challenge in drug-naive mice increases Ser133 cAMP response element-binding protein (CREB) phosphorylation in the NAc via Ca(v)1.3 channels and via a dopamine D(1)-dependent mechanism, independent of the extracellular signal-regulated kinase (ERK) pathway, an important mediator of psychostimulant-induced plasticity. In contrast, in amphetamine- and cocaine-preexposed mice, an amphetamine or cocaine challenge no longer activates CREB unless Ca(v)1.2 LTCCs are blocked. This Ca(v)1.2-dependent blunting of CREB activation that underlies expression of locomotor sensitization occurs only after extended drug-free periods and involves recruitment of D(1) receptors and the ERK pathway. Thus, our results demonstrate that specific LTCC subunits are required for the development (Ca(v)1.3) versus expression (Ca(v)1.2) of psychostimulant sensitization and that subunit-specific signaling pathways recruited by psychostimulants underlies long-term drug-induced behavioral responses.
Asunto(s)
Anfetamina/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Cocaína/farmacología , Actividad Motora/efectos de los fármacos , Nifedipino/farmacología , Núcleo Accumbens/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Aminoacetonitrilo/análogos & derivados , Aminoacetonitrilo/farmacología , Anfetamina/antagonistas & inhibidores , Animales , Benzazepinas/farmacología , Canales de Calcio Tipo L/genética , Estimulantes del Sistema Nervioso Central/antagonistas & inhibidores , Cocaína/antagonistas & inhibidores , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Interacciones Farmacológicas , Masculino , Ratones , Ratones Noqueados , Actividad Motora/genética , Núcleo Accumbens/metabolismoRESUMEN
We studied wild-type (WT) and Cav1.3(-/-) mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca(2+) channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca(2+) buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivating BK currents sustained mainly the afterhyperpolarization of the short APs (2-3 ms) and only partially the pacemaker current during the long interspike (300-500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca(2+) current and the corresponding Ca(2+)-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.
Asunto(s)
Médula Suprarrenal/citología , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/metabolismo , Células Cromafines/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Corteza Suprarrenal/metabolismo , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Biofisica , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Células Cultivadas , Quelantes/farmacología , Células Cromafines/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Estimulación Eléctrica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Indoles/farmacología , Ionóforos/farmacología , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Araña/farmacología , Factores de TiempoRESUMEN
Degeneration of dopaminergic neurons in the substantia nigra causes the motor symptoms of Parkinson's disease. The mechanisms underlying this age-dependent and region-selective neurodegeneration remain unclear. Here we identify Cav2.3 channels as regulators of nigral neuronal viability. Cav2.3 transcripts were more abundant than other voltage-gated Ca2+ channels in mouse nigral neurons and upregulated during aging. Plasmalemmal Cav2.3 protein was higher than in dopaminergic neurons of the ventral tegmental area, which do not degenerate in Parkinson's disease. Cav2.3 knockout reduced activity-associated nigral somatic Ca2+ signals and Ca2+-dependent after-hyperpolarizations, and afforded full protection from degeneration in vivo in a neurotoxin Parkinson's mouse model. Cav2.3 deficiency upregulated transcripts for NCS-1, a Ca2+-binding protein implicated in neuroprotection. Conversely, NCS-1 knockout exacerbated nigral neurodegeneration and downregulated Cav2.3. Moreover, NCS-1 levels were reduced in a human iPSC-model of familial Parkinson's. Thus, Cav2.3 and NCS-1 may constitute potential therapeutic targets for combatting Ca2+-dependent neurodegeneration in Parkinson's disease.
Asunto(s)
Envejecimiento/genética , Canales de Calcio Tipo R/genética , Proteínas de Transporte de Catión/genética , Supervivencia Celular/genética , Neuronas Dopaminérgicas/metabolismo , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Enfermedad de Parkinson/genética , Envejecimiento/metabolismo , Animales , Canales de Calcio Tipo R/metabolismo , Señalización del Calcio , Proteínas de Transporte de Catión/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Ratones Noqueados , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Sustancia Negra/metabolismo , Sustancia Negra/patología , Regulación hacia Arriba , Área Tegmental Ventral/metabolismo , Área Tegmental Ventral/patologíaRESUMEN
The spontaneous activity of sinoatrial node (SAN) pacemaker cells is generated by a functional interplay between the activity of ionic currents of the plasma membrane and intracellular Ca2+ dynamics. The molecular correlate of a dihydropyridine (DHP)-sensitive sustained inward Na+ current (I st), a key player in SAN automaticity, is still unknown. Here we show that I st and the L-type Ca2+ current (I Ca,L) share CaV1.3 as a common molecular determinant. Patch-clamp recordings of mouse SAN cells showed that I st is activated in the diastolic depolarization range, and displays Na+ permeability and minimal inactivation and sensitivity to I Ca,L activators and blockers. Both CaV1.3-mediated I Ca,L and I st were abolished in CaV1.3-deficient (CaV1.3-/-) SAN cells but the CaV1.2-mediated I Ca,L current component was preserved. In SAN cells isolated from mice expressing DHP-insensitive CaV1.2 channels (CaV1.2DHP-/-), I st and CaV1.3-mediated I Ca,L displayed overlapping sensitivity and concentration-response relationships to the DHP blocker nifedipine. Consistent with the hypothesis that CaV1.3 rather than CaV1.2 underlies I st, a considerable fraction of I Ca,L was resistant to nifedipine inhibition in CaV1.2DHP-/- SAN cells. These findings identify CaV1.3 channels as essential molecular components of the voltage-dependent, DHP-sensitive I st Na+ current in the SAN.
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Potenciales de Acción/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Dihidropiridinas/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Potenciales de Acción/genética , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Células Cultivadas , Frecuencia Cardíaca/genética , Isradipino/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Nifedipino/farmacología , Técnicas de Placa-Clamp , Nodo Sinoatrial/citología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/metabolismoRESUMEN
Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata-like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel's inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10-4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%-1.9%] versus 0.4% [0.3%-0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.
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Adenoma , Neoplasias de las Glándulas Suprarrenales , Glándulas Suprarrenales/patología , Aldosterona/metabolismo , Canales de Calcio Tipo L/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Hiperaldosteronismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Adenoma/genética , Adenoma/patología , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/patología , Adulto , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/patología , Masculino , Persona de Mediana EdadRESUMEN
AIMS: Sino-atrial node (SAN) automaticity is an essential mechanism of heart rate generation that is still not completely understood. Recent studies highlighted the importance of intracellular Ca(2+) ([Ca(2+)]i) dynamics during SAN pacemaker activity. Nevertheless, the functional role of voltage-dependent L-type Ca(2+) channels in controlling SAN [Ca(2+)]i release is largely unexplored. Since Cav1.3 is the predominant L-type Ca(2+) channel isoform in SAN cells, we studied [Ca(2+)]i dynamics in isolated cells and ex vivo SAN preparations explanted from wild-type (WT) and Cav1.3 knockout (KO) mice (Cav1.3(-/-)). METHODS AND RESULTS: We found that Cav1.3 deficiency strongly impaired [Ca(2+)]i dynamics, reducing the frequency of local [Ca(2+)]i release events and preventing their synchronization. This impairment inhibited the generation of Ca(2+) transients and delayed spontaneous activity. We also used action potentials recorded in WT SAN cells as voltage-clamp commands for Cav1.3(-/-) cells. Although these experiments showed abolished Ca(2+) entry through L-type Ca(2+) channels in the diastolic depolarization range of KO SAN cells, their sarcoplasmic reticulum Ca(2+) load remained normal. ß-Adrenergic stimulation enhanced pacemaking of both genotypes, though, Cav1.3(-/-) SAN cells remained slower than WT. Conversely, we rescued pacemaker activity in Cav1.3(-/-) SAN cells and intact tissues through caffeine-mediated stimulation of Ca(2+)-induced Ca(2+) release. CONCLUSIONS: Cav1.3 channels play a critical role in the regulation of [Ca(2+)]i dynamics, providing an unanticipated mechanism for triggering local [Ca(2+)]i releases and thereby controlling pacemaker activity. Our study also provides an additional pathophysiological mechanism for congenital SAN dysfunction and heart block linked to Cav1.3 loss of function in humans.
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Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Retículo Sarcoplasmático/metabolismo , Potenciales de Acción/fisiología , Animales , Canales de Calcio Tipo L/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Marcapaso Artificial , Canal Liberador de Calcio Receptor de Rianodina/genética , Nodo Sinoatrial/metabolismoRESUMEN
Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, afferent synaptic transmission is triggered by spontaneous Ca2+ spikes of IHCs, which are under efferent cholinergic control. Around the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired, and Ca2+ spikes as well as the cholinergic innervation are lost. Here, we performed patch-clamp measurements in IHCs of mice lacking the CaV1.3 channel (CaV1.3-/-) to investigate the role of this prevailing voltage-gated Ca2+ channel in IHC development and synaptic function. The small Ca2+ current remaining in IHCs from 3-week-old CaV1.3-/- mice was mainly mediated by L-type Ca2+ channels, because it was sensitive to dihydropyridines but resistant to inhibitors of non-L-type Ca2+ channels such as omega-conotoxins GVIA and MVIIC and SNX-482. Depolarization induced only marginal exocytosis in CaV1.3-/- IHC, which was solely mediated by L-type Ca2+ channels, whereas robust exocytic responses were elicited by photolysis of caged Ca2+. Secretion triggered by short depolarizations was reduced proportionally to the Ca2+ current, suggesting that the coupling of the remaining channels to exocytosis was unchanged. CaV1.3-/- IHCs lacked the Ca2+ action potentials and displayed a complex developmental failure. Most strikingly, we observed a continued presence of efferent cholinergic synaptic transmission and a lack of functional large-conductance Ca2+-activated K+ channels up to 4 weeks after birth. We conclude that CaV1.3 channels are essential for normal hair cell development and synaptic transmission.
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Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo L/metabolismo , Cóclea/citología , Cóclea/fisiología , Células Ciliadas Auditivas Internas/metabolismo , Envejecimiento/fisiología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/genética , Separación Celular , Sordera/genética , Capacidad Eléctrica , Exocitosis/fisiología , Células Ciliadas Auditivas Internas/citología , Canales de Potasio de Gran Conductancia Activados por el Calcio , Potenciales de la Membrana/fisiología , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Canales de Potasio Calcio-Activados/metabolismoRESUMEN
The preferential degeneration of Substantia nigra dopamine midbrain neurons (SN DA) causes the motor-symptoms of Parkinson's disease (PD). Voltage-gated L-type calcium channels (LTCCs), especially the Cav1.3-subtype, generate an activity-related oscillatory Ca(2+) burden in SN DA neurons, contributing to their degeneration and PD. While LTCC-blockers are already in clinical trials as PD-therapy, age-dependent functional roles of Cav1.3 LTCCs in SN DA neurons remain unclear. Thus, we analysed juvenile and adult Cav1.3-deficient mice with electrophysiological and molecular techniques. To unmask compensatory effects, we compared Cav1.3 KO mice with pharmacological LTCC-inhibition. LTCC-function was not necessary for SN DA pacemaker-activity at either age, but rather contributed to their pacemaker-precision. Moreover, juvenile Cav1.3 KO but not WT mice displayed adult wildtype-like, sensitised inhibitory dopamine-D2-autoreceptor (D2-AR) responses that depended upon both, interaction of the neuronal calcium sensor NCS-1 with D2-ARs, and on voltage-gated T-type calcium channel (TTCC) activity. This functional KO-phenotype was accompanied by cell-specific up-regulation of NCS-1 and Cav3.1-TTCC mRNA. Furthermore, in wildtype we identified an age-dependent switch of TTCC-function from contributing to SN DA pacemaker-precision in juveniles to pacemaker-frequency in adults. This novel interplay of Cav1.3 L-type and Cav3.1 T-type channels, and their modulation of SN DA activity-pattern and D2-AR-sensitisation, provide new insights into flexible age- and calcium-dependent activity-control of SN DA neurons and its pharmacological modulation.
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Autorreceptores/metabolismo , Canales de Calcio Tipo L/deficiencia , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/metabolismo , Neuronas Dopaminérgicas/metabolismo , Receptores de Dopamina D2/metabolismo , Sustancia Negra/metabolismo , Factores de Edad , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Regulación de la Expresión Génica , Masculino , Potenciales de la Membrana , Ratones , Ratones Noqueados , Proteínas Sensoras del Calcio Neuronal/genética , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/genética , Neuropéptidos/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.
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Enfermedades de la Corteza Suprarrenal/genética , Canales de Calcio Tipo L/genética , Hipertensión/genética , Mutación , ATPasa Intercambiadora de Sodio-Potasio/genética , Enfermedades de la Corteza Suprarrenal/complicaciones , Enfermedades de la Corteza Suprarrenal/diagnóstico , Sustitución de Aminoácidos , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Análisis por Conglomerados , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Perfilación de la Expresión Génica , Humanos , Hipertensión/diagnóstico , Hipertensión/etiología , Masculino , Conformación Proteica , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
AMPA receptor (AMPAR) plasticity at glutamatergic synapses in the mesostriatal dopaminergic pathway has been implicated in persistent cocaine-induced behavioral responses; however, the precise mechanism underlying these changes remains unknown. Utilizing cocaine psychomotor sensitization in mice we find that repeated cocaine results in a basal reduction of Ser 845 GluA1 and cell surface GluA1 levels in the dorsal striatum (dStr) following a protracted withdrawal period, an adaptation that is dependent on Cav 1.3 channels but not those expressed in the VTA. We find that the basally-induced decrease in this phosphoprotein is the result of recruitment of the striatal dopamine D2 pathway, as evidenced by enhanced levels of D2 receptor (D2R) mRNA expression and D2R function as examined using the D2R antagonist, eticlopride, as well as alterations in the phosphorylation status of several downstream molecular targets of D2R's, including CREB, DARPP-32, Akt and GSK3ß. Taken together with our recently published findings examining similar phenomena in the nucleus accumbens (NAc), these results underscore the utilization of divergent molecular mechanisms in the dStr, in mediating cocaine-induced persistent behavioral changes.
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Canales de Calcio Tipo L/fisiología , Cocaína/farmacología , Inhibidores de Captación de Dopamina/farmacología , Receptores AMPA/fisiología , Receptores de Dopamina D2/fisiología , Adaptación Fisiológica , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiología , Técnicas de Silenciamiento del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , ARN Mensajero/metabolismo , Síndrome de Abstinencia a Sustancias/fisiopatologíaRESUMEN
BACKGROUND: Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block. METHODS AND RESULTS: We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca(2+) current (I(Ca,L)) densities and positive (rightward) voltage shifts in I(Ca,L) activation. Pacemaker current (I(f)) was not affected by CIO. Because I(Ca,L) in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of Ca(V)1.3 channels (in addition to Ca(V)1.2 channels), our data suggest that elevated iron preferentially suppresses Ca(V)1.3 channel function. Consistent with this suggestion, CIO reduced Ca(V)1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in Ca(V)1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in Ca(V)1.3 knockout mice. CONCLUSIONS: Our results demonstrate that CIO selectively reduces Ca(V)1.3-mediated I(Ca,L), leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.
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Fibrilación Atrial/fisiopatología , Bradicardia/fisiopatología , Canales de Calcio Tipo L/fisiología , Sistema de Conducción Cardíaco/fisiopatología , Hierro/efectos adversos , Animales , Fibrilación Atrial/etiología , Bradicardia/etiología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/genética , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sistema de Conducción Cardíaco/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Hierro/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Nodo Sinoatrial/efectos de los fármacos , Nodo Sinoatrial/fisiopatologíaRESUMEN
The family of voltage-gated calcium channels serves as the key transducers of cell surface membrane potential changes into local intracellular calcium transients that initiate many different physiological events. There are 10 members of the voltage-gated calcium channel family that have been characterized in mammals, and they serve distinct roles in cellular signal transduction. This article presents the molecular relationships and physiological functions of these calcium channel proteins and provides comprehensive information on their molecular, genetic, physiological, and pharmacological properties.