RESUMEN
The neuronal-type (N-type) voltage-gated calcium (Cav) channels, which are designated Cav2.2, have an important role in the release of neurotransmitters1-3. Ziconotide is a Cav2.2-specific peptide pore blocker that has been clinically used for treating intractable pain4-6. Here we present cryo-electron microscopy structures of human Cav2.2 (comprising the core α1 and the ancillary α2δ-1 and ß3 subunits) in the presence or absence of ziconotide. Ziconotide is thoroughly coordinated by helices P1 and P2, which support the selectivity filter, and the extracellular loops (ECLs) in repeats II, III and IV of α1. To accommodate ziconotide, the ECL of repeat III and α2δ-1 have to tilt upward concertedly. Three of the voltage-sensing domains (VSDs) are in a depolarized state, whereas the VSD of repeat II exhibits a down conformation that is stabilized by Cav2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Cav2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Cav channels.
Asunto(s)
Analgésicos no Narcóticos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Microscopía por Crioelectrón , omega-Conotoxinas/farmacología , Canales de Calcio Tipo N/ultraestructura , Humanos , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacología , Conformación Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacosRESUMEN
Migraine is a common neurological disease that affects about 11% of the adult population. The disease is divided into two main clinical subtypes: migraine with aura and migraine without aura. According to the neurovascular theory of migraine, the activation of the trigeminovascular system (TGVS) and the release of numerous neuropeptides, including calcitonin gene-related peptide (CGRP) are involved in headache pathogenesis. TGVS can be activated by cortical spreading depression (CSD), a phenomenon responsible for the aura. The mechanism of CSD, stemming in part from aberrant interactions between neurons and glia have been studied in models of familial hemiplegic migraine (FHM), a rare monogenic form of migraine with aura. The present review focuses on those interactions, especially as seen in FHM type 1, a variant of the disease caused by a mutation in CACNA1A, which encodes the α1A subunit of the P/Q-type voltage-gated calcium channel.
Asunto(s)
Canales de Calcio/metabolismo , Trastornos Migrañosos/etiología , Neuroglía/patología , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Humanos , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/fisiopatología , Mutación , Neuroglía/metabolismoRESUMEN
Sound information encoding within the initial synapses in the auditory brainstem requires reliable and precise synaptic transmission in response to rapid and large fluctuations in action potential (AP) firing rates. The magnitude and location of Ca2+ entry through voltage-gated Ca2+ channels (CaV) in the presynaptic terminal are key determinants in triggering AP-mediated release. In the mammalian central nervous system (CNS), the CaV2.1 subtype is the critical subtype for CNS function, since it is the most efficient CaV2 subtype in triggering AP-mediated synaptic vesicle (SV) release. Auditory brainstem synapses utilize CaV2.1 to sustain fast and repetitive SV release to encode sound information. Therefore, understanding the presynaptic mechanisms that control CaV2.1 localization, organization and biophysical properties are integral to understanding auditory processing. Here, we review our current knowledge about the control of presynaptic CaV2 abundance and organization in the auditory brainstem and impact on the regulation of auditory processing.
Asunto(s)
Tronco Encefálico/fisiología , Canales de Calcio Tipo N/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Activación del Canal Iónico/fisiología , Proteínas del Tejido Nervioso/fisiología , Terminales Presinápticos/fisiología , Animales , Vías Auditivas/fisiología , Calcio/metabolismo , Canales de Calcio Tipo N/química , Humanos , Transporte Iónico , Mamíferos/fisiología , Proteínas del Tejido Nervioso/química , Dominios Proteicos , Subunidades de Proteína , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismoRESUMEN
CACNA1A pathogenic variants have been linked to several neurological disorders including familial hemiplegic migraine and cerebellar conditions. More recently, de novo variants have been associated with severe early onset developmental encephalopathies. CACNA1A is highly expressed in the central nervous system and encodes the pore-forming CaVα1 subunit of P/Q-type (Cav2.1) calcium channels. We have previously identified a patient with a de novo missense mutation in CACNA1A (p.Y1384C), characterized by hemiplegic migraine, cerebellar atrophy and developmental delay. The mutation is located at the transmembrane S5 segment of the third domain. Functional analysis in two predominant splice variants of the neuronal Cav2.1 channel showed a significant loss of function in current density and changes in gating properties. Moreover, Y1384 variants exhibit differential splice variant-specific effects on recovery from inactivation. Finally, structural analysis revealed structural damage caused by the tyrosine substitution and changes in electrostatic potentials.
Asunto(s)
Canales de Calcio Tipo N/genética , Cerebelo/patología , Discapacidades del Desarrollo/genética , Predisposición Genética a la Enfermedad , Migraña con Aura/genética , Mutación/genética , Adolescente , Adulto , Empalme Alternativo/genética , Atrofia , Fenómenos Biofísicos , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Línea Celular , Preescolar , Discapacidades del Desarrollo/complicaciones , Femenino , Humanos , Recién Nacido , Activación del Canal Iónico , Masculino , Migraña con Aura/complicaciones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Isoformas de Proteínas/genética , Estructura Secundaria de Proteína , Homología Estructural de ProteínaRESUMEN
The dominant role of CaV2 voltage-gated calcium channels for driving neurotransmitter release is broadly conserved. Given the overlapping functional properties of CaV2 and CaV1 channels, and less so CaV3 channels, it is unclear why there have not been major shifts toward dependence on other CaV channels for synaptic transmission. Here, we provide a structural and functional profile of the CaV2 channel cloned from the early-diverging animal Trichoplax adhaerens, which lacks a nervous system but possesses single gene homologues for CaV1-CaV3 channels. Remarkably, the highly divergent channel possesses similar features as human CaV2.1 and other CaV2 channels, including high voltage-activated currents that are larger in external Ba2+ than in Ca2+; voltage-dependent kinetics of activation, inactivation, and deactivation; and bimodal recovery from inactivation. Altogether, the functional profile of Trichoplax CaV2 suggests that the core features of presynaptic CaV2 channels were established early during animal evolution, after CaV1 and CaV2 channels emerged via proposed gene duplication from an ancestral CaV1/2 type channel. The Trichoplax channel was relatively insensitive to mammalian CaV2 channel blockers ω-agatoxin-IVA and ω-conotoxin-GVIA and to metal cation blockers Cd2+ and Ni2+ Also absent was the capacity for voltage-dependent G-protein inhibition by co-expressed Trichoplax Gßγ subunits, which nevertheless inhibited the human CaV2.1 channel, suggesting that this modulatory capacity evolved via changes in channel sequence/structure, and not G proteins. Last, the Trichoplax channel was immunolocalized in cells that express an endomorphin-like peptide implicated in cell signaling and locomotive behavior and other likely secretory cells, suggesting contributions to regulated exocytosis.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Señalización del Calcio , Calcio/metabolismo , Activación del Canal Iónico , Transmisión Sináptica , Secuencia de Aminoácidos , Animales , Cadmio/farmacología , Níquel/farmacología , Filogenia , Placozoa , Homología de Secuencia de AminoácidoRESUMEN
Arginine vasopressin (AVP) neurons play essential roles in sensing the change in systemic osmolarity and regulating AVP release from their neuronal terminals to maintain the plasma osmolarity. AVP exocytosis depends on the Ca2+ entry via voltage-gated Ca2+ channels (VGCCs) in AVP neurons. In this study, suppression by siRNA-mediated knockdown and pharmacological sensitivity of VGCC currents evidenced molecular and functional expression of N-type Cav2.2 and T-type Cav3.1 in AVP neurons under normotonic conditions. Also, both the Cav2.2 and Cav3.1 currents were found to be sensitive to flufenamic acid (FFA). TTX-insensitive spontaneous action potentials were suppressed by FFA and T-type VGCC blocker Ni2+. However, Cav2.2-selective ω-conotoxin GVIA failed to suppress the firing activity. Taken together, it is concluded that Cav2.2 and Cav3.1 are molecularly and functionally expressed and both are sensitive to FFA in unstimulated rat AVP neurons. Also, it is suggested that Cav3.1 is primarily involved in their action potential generation.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo T/metabolismo , Neuronas/metabolismo , Vasopresinas/metabolismo , Potenciales de Acción , Animales , Animales Modificados Genéticamente , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo T/química , Canales de Calcio Tipo T/genética , Señalización del Calcio , Masculino , Ratas , Ratas WistarRESUMEN
Presynaptic CaV 2.2 (N-type) channels are fundamental for transmitter release across the nervous system. The gene encoding CaV 2.2 channels, Cacna1b, contains alternatively spliced exons that result in functionally distinct splice variants (e18a, e24a, e31a, and 37a/37b). Alternative splicing of the cassette exon 18a generates two mRNA transcripts (+e18a-Cacna1b and ∆e18a-Cacna1b). In this study, using novel mouse genetic models and in situ hybridization (BaseScope™), we confirmed that +e18a-Cacna1b splice variants are expressed in monoaminergic regions of the midbrain. We expanded these studies and identified +e18a-Cacna1b mRNA in deep cerebellar cells and spinal cord motor neurons. Furthermore, we determined that +e18a-Cacna1b is enriched in cholecystokinin-expressing interneurons. Our results provide key information to understand cell-specific functions of CaV 2.2 channels.
Asunto(s)
Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Citoplasma/genética , Empalme Alternativo/genética , Animales , Sistema Nervioso Central/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Especificidad de Órganos , ARN Mensajero/genéticaRESUMEN
As an ω-conopeptide originally discovered from Conus striatus, SO-3 contains 25 amino acid residues and three disulfide bridges. Our previous study has shown that this peptide possesses potent analgesic activity in rodent pain models (mouse and rat), and it specifically inhibits an N-type calcium ion channel (Cav2.2). In the study presented here, we investigated the key amino acid residues for their inhibitory activity against Cav2.2 expressed in HEK 293 cells and analgesic activity in mice. To improve the inhibitory activity of SO-3, we also evaluated the effects of some amino acid residues derived from the corresponding residues of ω-peptide MVIIA, CVID, or GVIA. Our data reveal that Lys6, Ile11, and Asn14 are the important functional amino acid residues for SO-3. The replacement of some amino acid residues of SO-3 in loop 1 with the corresponding residues of CVID and GVIA improved the inhibitory activity of SO-3. The binding mode of Cav2.2 with SO-3 amino acids in loop 1 and loop 2 may be somewhat different from that of MVIIA. This study expanded our knowledge of the structure-activity relationship of ω-peptides and provided a new strategy for improving the potency of Cav2.2 inhibitors.
Asunto(s)
Analgésicos/farmacología , Conducta Animal/efectos de los fármacos , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Dolor/tratamiento farmacológico , Péptidos/farmacología , Analgésicos/química , Animales , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Dolor/metabolismo , Péptidos/química , Conformación Proteica , Ratas , Relación Estructura-ActividadRESUMEN
Both N- and T-type calcium ion channels have been implicated in pain transmission and the N-type channel is a well-validated target for the treatment of neuropathic pain. An SAR investigation of a series of substituted aminobenzothiazoles identified a subset of five compounds with comparable activity to the positive control Z160 in a FLIPR-based intracellular calcium response assay measuring potency at both CaV2.2 and CaV3.2 channels. These compounds may form the basis for the development of drug leads and tool compounds for assessing in vivo effects of variable modulation of CaV2.2 and CaV3.2 channels.
Asunto(s)
Bencimidazoles/síntesis química , Benzotiazoles/síntesis química , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo T/química , Ciclopropanos/síntesis química , Naftalenos/síntesis química , Piperidinas/síntesis química , Bencimidazoles/química , Bencimidazoles/farmacología , Benzotiazoles/química , Benzotiazoles/farmacología , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/química , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo T/efectos de los fármacos , Ciclopropanos/química , Ciclopropanos/farmacología , Estructura Molecular , Naftalenos/química , Naftalenos/farmacología , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacología , Piperidinas/química , Piperidinas/farmacología , Relación Estructura-ActividadRESUMEN
CaV channels are transmembrane proteins that mediate and regulate ion fluxes across cell membranes, and they are activated in response to action potentials to allow Ca2+ influx. Since ion channels are composed of charge or polar groups, an external alternating electric field may affect the ion-selective membrane transport and the performance of the channel. In this article, we have investigated the effect of an external GHz electric field on the dynamics of calcium ions in the selectivity filter of the CaV Ab channel. Molecular dynamics (MD) simulations and the potential of mean force (PMF) calculations were carried out, via the umbrella sampling method, to determine the free energy profile of Ca2+ ions in the CaV Ab channels in presence and absence of an external field. Exposing CaV Ab channel to 1, 2, 3, 4, and 5 GHz electric fields increases the depth of the potential energy well and this may result in an increase in the affinity and strength of Ca2+ ions to binding sites in the selectivity filter the channel. This increase of strength of Ca2+ ions binding in the selectivity filter may interrupt the mechanism of Ca2+ ion conduction, and leads to a reduction of Ca2+ ion permeation through the CaV Ab channel.
Asunto(s)
Arcobacter/metabolismo , Proteínas Bacterianas/metabolismo , Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Arcobacter/química , Proteínas Bacterianas/química , Calcio/química , Canales de Calcio Tipo N/química , Cationes Bivalentes/química , Cationes Bivalentes/metabolismo , Electricidad , Transporte Iónico , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , TermodinámicaRESUMEN
Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand-like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Calmodulina/metabolismo , Empalme Alternativo , Animales , Sitios de Unión , Calcio/metabolismo , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Células HEK293 , Humanos , Potenciales de la Membrana , Unión Proteica , RatasRESUMEN
Type two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability. 1 Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3. 2 We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons. 3 In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or ß4 alone failed to increase current density, suggesting that the calcium current ceiling when CaV2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native CaV2.2 by recombinant channels. Both α2δ-1 and α2δ-2 seemed to promote CaV2.2 displacement by recombinant channel expression, while α2δ-3 appeared to protect CaV2.2 from displacement. Thus, we observe a selective prioritization of CaV channel functional expression in neurons by specific α2δ subunits. These data highlight a new function for α2δ subtypes that could shed light on subtype selectivity of CaV2 membrane expression.
Asunto(s)
Canales de Calcio Tipo N/biosíntesis , Neuronas/metabolismo , Subunidades de Proteína/metabolismo , Animales , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Masculino , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Ratas , Ratas WistarRESUMEN
Protein kinase C (PKC) isozymes modulate voltage-gated calcium (Cav) currents through Cav2.2 and Cav2.3 channels by targeting serine/threonine (Ser/Thr) phosphorylation sites of Cavα1 subunits. Stimulatory (Thr-422, Ser-2108 and Ser-2132) and inhibitory (Ser-425) sites were identified in the Cav2.2α1 subunits to PKCs ßII and ε. In the current study, we investigated if the homologous sites of Cav2.3α1 subunits (stimulatory: Thr-365, Ser-1995 and Ser-2011; inhibitory: Ser-369) behaved in similar manner. Several Ala and Asp mutants were constructed in Cav2.3α1 subunits in such a way that the Ser/Thr sites can be examined in isolation. These mutants or WT Cav2.3α1 along with auxiliary ß1b and α2/δ subunits were expressed in Xenopus oocytes and the effects of PKCs ßII and ε studied on the barium current (IBa). Among these sites, stimulatory Thr-365 and Ser-1995 and inhibitory Ser-369 behaved similar to their homologs in Cav2.2α1 subunits. Furthermore PKCs produced neither stimulation nor inhibition when stimulatory Thr-365 or Ser-1995 and inhibitory Ser-369 were present together. However, the PKCs potentiated the IBa when two stimulatory sites, Thr-365 and Ser-1995 were present together, thus overcoming the inhibitory effect of Ser-369. Taken together net PKC effect may be the difference between the responses of the stimulatory and inhibitory sites.
Asunto(s)
Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos , Isoenzimas/química , Isoenzimas/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Subunidades de Proteína , Serina/química , Serina/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Treonina/química , Treonina/metabolismo , Xenopus laevisRESUMEN
The ß subunit of voltage-gated Ca2+ (CaV) channels plays an important role in regulating gating of the α1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular localization of the CaV ß subunit is critical for this effect; N-terminal-dependent membrane targeting of the ß subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the ß subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a ß subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a ß subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The ß2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the ß subunit acts as an important regulator of CaV channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Activación del Canal Iónico , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Sitios de Unión , Canales de Calcio Tipo N/química , Células HEK293 , Humanos , Ratones , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , RatasRESUMEN
To analyze structural features of ω-Aga IVA, a gating modifier toxin from spider venom, we here investigated the NMR solution structure of ω-Aga IVA within DPC micelles. Under those conditions, the Cys-rich central region of ω-Aga IVA still retains the inhibitor Cys knot motif with three short antiparallel ß-strands seen in water. However, 15N HSQC spectra of ω-Aga IVA within micelles revealed that there are radical changes to the toxin's C-terminal tail and several loops upon binding to micelles. The C-terminal tail of ω-Aga IVA appears to assume a ß-turn like conformation within micelles, though it is disordered in water. Whole-cell patch clamp studies with several ω-Aga IVA analogs indicate that both the hydrophobic C-terminal tail and an Arg patch in the core region of ω-Aga IVA are critical for Cav2.1 blockade. These results suggest that the membrane environment stabilizes the structure of the toxin, enabling it to act in a manner similar to other gating modifier toxins, though its mode of interaction with the membrane and the channel is unique.
Asunto(s)
Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/ultraestructura , Membrana Celular/química , Membrana Dobles de Lípidos/química , Células de Purkinje/química , omega-Agatoxina IVA/química , Animales , Sitios de Unión , Conformación Molecular , Unión Proteica , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease characterized by loss of Purkinje cells in the cerebellum. SCA6 is caused by CAG trinucleotide repeat expansion in CACNA1A, which encodes Cav2.1, α1A subunit of P/Q-type calcium channel. However, the pathogenic mechanism and effective therapeutic treatments are still unknown. Here, we have succeeded in generating differentiated Purkinje cells that carry patient genes by combining disease-specific iPSCs and self-organizing culture technologies. Patient-derived Purkinje cells exhibit increased levels of full-length Cav2.1 protein but decreased levels of its C-terminal fragment and downregulation of the transcriptional targets TAF1 and BTG1. We further demonstrate that SCA6 Purkinje cells exhibit thyroid hormone depletion-dependent degeneration, which can be suppressed by two compounds, thyroid releasing hormone and Riluzole. Thus, we have constructed an in vitro disease model recapitulating both ontogenesis and pathogenesis. This model may be useful for pathogenic investigation and drug screening.
Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Células de Purkinje/patología , Ataxias Espinocerebelosas/patología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Dominios Proteicos , Células de Purkinje/efectos de los fármacos , Células de Purkinje/metabolismo , Riluzol/farmacología , Tirotropina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
At fast-transmitting presynaptic terminals Ca(2+) enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca(2+) that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca(2+) nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca(2+) from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Activación del Canal Iónico , Unión Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Canales de Calcio Tipo N/química , Ratones , Modelos Neurológicos , Multimerización de Proteína , Estructura Terciaria de Proteína , Ratas , XenopusRESUMEN
T- and N-type calcium channels have known for relating to therapy of neuropathic pain which is chronic, debilitating pain state. Neuropathic pain is caused by damage of the somatosensory system. It may be associated with abnormal sensations and pain produced by normally non-painful stimuli (allodynia). Neuropathic pain is very difficult to treat, and only some 40-60% of patients achieve partial relief. For a neuropathic pain therapy, anticonvulsant like Lamotrigine, Carbamazepine and a topical anesthetic such as Lidocaine are used. We synthesized 15 novel amine derivatives and evaluated their activities against T-type and N-type calcium channels by whole-cell patch clamp recording on HEK293 cells. Among the tested compounds, compound 10 showed good inhibitory activity for both T-type and N-type calcium channels with the IC50 value of 1.9 µM and 4.3 µM, respectively. Compound 10 also showed good analgesic activity on rat spinal cord injury model.
Asunto(s)
Aminas/química , Bloqueadores de los Canales de Calcio/química , Aminas/farmacología , Aminas/uso terapéutico , Animales , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo T/química , Canales de Calcio Tipo T/metabolismo , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Actividad Motora/efectos de los fármacos , Neuralgia/tratamiento farmacológico , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel ß-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-ß-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.