RESUMO
Presynaptic voltage-gated Ca2+ channel (CaV ) subtype abundance at mammalian synapses regulates synaptic transmission in health and disease. In the mammalian central nervous system (CNS), most presynaptic terminals are CaV 2.1 dominant with a developmental reduction in CaV 2.2 and CaV 2.3 levels, and CaV 2 subtype levels are altered in various diseases. However, the molecular mechanisms controlling presynaptic CaV 2 subtype levels are largely unsolved. Because the CaV 2 α1 subunit cytoplasmic regions contain varying levels of sequence conservation, these regions are proposed to control presynaptic CaV 2 subtype preference and abundance. To investigate the potential role of these regions, we expressed chimeric CaV 2.1 α1 subunits containing swapped motifs with the CaV 2.2 and CaV 2.3 α1 subunit on a CaV 2.1/CaV 2.2 null background at the calyx of Held presynaptic terminals. We found that expression of CaV 2.1 α1 subunit chimeras containing the CaV 2.3 loop II-III region or cytoplasmic C-terminus (CT) resulted in a large reduction of presynaptic Ca2+ currents compared to the CaV 2.1 α1 subunit. However, the Ca2+ current sensitivity to the CaV 2.1 blocker agatoxin-IVA was the same between the chimeras and the CaV 2.1 α1 subunit. Additionally, we found no reduction in presynaptic Ca2+ currents with CaV 2.1/2.2 cytoplasmic CT chimeras. We conclude that the motifs in the CaV 2.1 loop II-III and CT do not individually regulate CaV 2.1 preference, although these motifs control CaV 2.1 levels and the CaV 2.3 CT contains motifs that negatively regulate presynaptic CaV 2.3 levels. We propose that the motifs controlling presynaptic CaV 2.1 preference are distinct from those regulating CaV 2.1 levels and may act synergistically to impact pathways regulating CaV 2.1 preference and abundance. KEY POINTS: Presynaptic CaV 2 subtype abundance regulates neuronal circuit properties, although the mechanisms regulating presynaptic CaV 2 subtype abundance and preference remain enigmatic. The CaV α1 subunit determines subtype and contains multiple motifs implicated in regulating presynaptic subtype abundance and preference. The CaV 2.1 α1 subunit domain II-III loop and cytoplasmic C-terminus are positive regulators of presynaptic CaV 2.1 abundance but do not regulate preference. The CaV 2.3 α1 subunit cytoplasmic C-terminus negatively regulates presynaptic CaV 2 subtype abundance but not preference, whereas the CaV 2.2 α1 subunit cytoplasmic C-terminus is not a key regulator of presynaptic CaV 2 subtype abundance or preference. The CaV 2 α1 subunit motifs determining the presynaptic CaV 2 preference are distinct from abundance.
Assuntos
Canais de Cálcio Tipo N , Transmissão Sináptica , Animais , Canais de Cálcio Tipo N/genética , Transmissão Sináptica/fisiologia , Sinapses/fisiologia , Terminações Pré-Sinápticas/fisiologia , Neurônios/metabolismo , Mamíferos/metabolismoRESUMO
The distance between CaV2.1 voltage-gated Ca2+ channels and the Ca2+ sensor responsible for vesicle release at presynaptic terminals is critical for determining synaptic strength. Yet, the molecular mechanisms responsible for a loose coupling configuration of CaV2.1 in certain synapses or developmental periods and a tight one in others remain unknown. Here, we examine the nanoscale organization of two CaV2.1 splice isoforms (CaV2.1[EFa] and CaV2.1[EFb]) at presynaptic terminals by superresolution structured illumination microscopy. We find that CaV2.1[EFa] is more tightly co-localized with presynaptic markers than CaV2.1[EFb], suggesting that alternative splicing plays a crucial role in the synaptic organization of CaV2.1 channels.
Assuntos
Terminações Pré-Sinápticas , Vesículas Sinápticas , Isoformas de Proteínas , SinapsesRESUMO
Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivoSIGNIFICANCE STATEMENT Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.
Assuntos
Canais de Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Cálcio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Cacna1a encodes the pore-forming α1A subunit of CaV2.1 voltage-dependent calcium channels, which regulate neuronal excitability and synaptic transmission. Purkinje cells in the cortex of cerebellum abundantly express these CaV2.1 channels. Here, we show that homozygous tottering (tg) mice, which carry a loss-of-function Cacna1a mutation, exhibit severely impaired learning in Pavlovian eyeblink conditioning, which is a cerebellar-dependent learning task. Performance of reflexive eyeblinks is unaffected in tg mice. Transient seizure activity in tg mice further corrupted the amplitude of eyeblink conditioned responses. Our results indicate that normal calcium homeostasis is imperative for cerebellar learning and that the oscillatory state of the brain can affect the expression thereof.NEW & NOTEWORTHY In this study, we confirm the importance of normal calcium homeostasis in neurons for learning and memory formation. In a mouse model with a mutation in an essential calcium channel that is abundantly expressed in the cerebellum, we found severely impaired learning in eyeblink conditioning. Eyeblink conditioning is a cerebellar-dependent learning task. During brief periods of brain-wide oscillatory activity, as a result of the mutation, the expression of conditioned eyeblinks was even further disrupted.
Assuntos
Piscadela , Canais de Cálcio Tipo N/genética , Condicionamento Clássico , Animais , Cálcio/metabolismo , Cerebelo/fisiologia , Feminino , Homozigoto , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Migraine patients often report (inter)ictal hypersensitivity to light, but the underlying mechanisms remain an enigma. Both hypo- and hyperresponsivity of the visual network have been reported, which may reflect either intra-individual dynamics of the network or large inter-individual variation in the measurement of human visual evoked potential data. Therefore, we studied visual system responsivity in freely behaving mice using combined epidural electroencephalography and intracortical multi-unit activity to reduce variation in recordings and gain insight into visual cortex dynamics. For better clinical translation, we investigated transgenic mice that carry the human pathogenic R192Q missense mutation in the α1A subunit of voltage-gated CaV 2.1 Ca2+ channels leading to enhanced neurotransmission and familial hemiplegic migraine type 1 in patients. Visual evoked potentials were studied in response to visual stimulation paradigms with flashes of light. Following intensity-dependent visual stimulation, FHM1 mutant mice displayed faster visual evoked potential responses, with lower initial amplitude, followed by less pronounced neuronal suppression compared to wild-type mice. Similar to what was reported for migraine patients, frequency-dependent stimulation in mutant mice revealed enhanced photic drive in the EEG beta-gamma band. The frequency-dependent increases in visual network responses in mutant mice may reflect the context-dependent enhancement of visual cortex excitability, which could contribute to our understanding of sensory hypersensitivity in migraine.
Assuntos
Enxaqueca com Aura , Animais , Canais de Cálcio Tipo N , Modelos Animais de Doenças , Potenciais Evocados Visuais , Humanos , Camundongos , Camundongos TransgênicosRESUMO
Calcium ions serve as an important intracellular messenger in many diverse pathways, ranging from excitation coupling in muscles to neurotransmitter release in neurons. Physiologically, the concentration of free intracellular Ca2+ is up to 10,000 times less than that of the extracellular concentration, and increases of 10- to 100-fold in intracellular Ca2+ are observed during signaling events. Voltage-gated calcium channels (VGCCs) located on the plasma membrane serve as one of the main ways in which Ca2+ is able to enter the cell. Given that Ca2+ functions as a ubiquitous intracellular messenger, it is imperative that VGCCs are under tight regulation to ensure that intracellular Ca2+ concentration remains within the physiological range. In this chapter, we explore VGCCs' inherent control of Ca2+ entry as well as the effects of alternative splicing in CaV2.1 and posttranslational modifications of CaV1.2/CaV1.3 such as phosphorylation and ubiquitination. Deviation from this physiological range will result in deleterious effects known as calcium channelopathies, some of which will be explored in this chapter.
Assuntos
Canais de Cálcio , Sinalização do Cálcio , Encéfalo/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Neurônios/metabolismoRESUMO
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.
Assuntos
Canais de Cálcio/metabolismo , Transtornos de Enxaqueca/etiologia , Neuroglia/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Humanos , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Mutação , Neuroglia/metabolismoRESUMO
The CACNA1A gene encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 Ca2+ channel, essential in neurotransmission, especially in Purkinje cells. Mutations in CACNA1A result in great clinical heterogeneity with progressive symptoms, paroxysmal events or both. During infancy, clinical and neuroimaging findings may be unspecific, and no dysmorphic features have been reported. We present the clinical, radiological and evolutionary features of three patients with congenital ataxia, one of them carrying a new variant. We report the structural localization of variants and their expected functional consequences. There was an improvement in cerebellar syndrome over time despite a cerebellar atrophy progression, inconsistent response to acetazolamide and positive response to methylphenidate. The patients shared distinctive facial gestalt: oval face, prominent forehead, hypertelorism, downslanting palpebral fissures and narrow nasal bridge. The two α1A affected residues are fully conserved throughout evolution and among the whole human CaV channel family. They contribute to the channel pore and the voltage sensor segment. According to structural data analysis and available functional characterization, they are expected to exert gain- (F1394L) and loss-of-function (R1664Q/R1669Q) effect, respectively. Among the CACNA1A-related phenotypes, our results suggest that non-progressive congenital ataxia is associated with developmental delay and dysmorphic features, constituting a recognizable syndromic neurodevelopmental disorder.
Assuntos
Ataxia/patologia , Canais de Cálcio/genética , Mutação , Adulto , Sequência de Aminoácidos , Ataxia/congênito , Ataxia/etiologia , Ataxia/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Criança , Feminino , Humanos , Masculino , Neuroimagem , Fenótipo , Conformação Proteica , Homologia de Sequência , Relação Estrutura-Atividade , Adulto JovemRESUMO
Human mutations in the CACNA1A gene that encodes the pore-forming α1A subunit of the voltage-gated CaV2.1 (P/Q-type) Ca2+ channel cause multiple neurological disorders including sporadic and familial hemiplegic migraine, as well as cerebellar pathologies such as episodic ataxia, progressive ataxia, and early-onset cerebellar syndrome consistent with the definition of congenital ataxia (CA), with presentation before the age of 2 years. Such a pathological role is in accordance with the physiological relevance of CaV2.1 in neuronal tissue, especially in the cerebellum. This review deals with the report of the main clinical features defining CA, along with the presentation of an increasing number of CACNA1A genetic variants linked to this severe cerebellar disorder in the context of Ca2+ homeostasis alteration. Moreover, the review describes each pathological mutation according to structural location and known molecular and cellular functional effects in both heterologous expression systems and animal models. In view of this information in correlation with the clinical phenotype, we take into consideration different pathomechanisms underlying the observed motor dysfunction in CA patients carrying CACNA1A mutations. Present therapeutic management in CA and options for the development of future personalized treatment based on CaV2.1 dysfunction are also discussed.
Assuntos
Ataxia/genética , Canais de Cálcio/genética , Mutação/genética , Sequência de Aminoácidos , Animais , HumanosRESUMO
Free Calcium (Ca2+) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca2+ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca2+ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca2+-permeable channels a major signalling tool of red blood cells. However, the internal Ca2+ concentration is tightly controlled, regulated and maintained primarily by the Ca2+ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca2+ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca2+ signals only recently it became evident, that a temporal increase in intracellular Ca2+ can also have positive effects such as the modulation of the red blood cells O2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca2+ channels and Ca2+-regulated channels in red blood cells, namely the Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca2+-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle. Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gárdos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.
Assuntos
Canais de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Eritrócitos/fisiologia , Doenças Hematológicas/fisiopatologia , Humanos , MutaçãoRESUMO
The regulation of insulin biosynthesis and secretion in pancreatic ß-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic ß-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Caseína Quinase II/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , RatosRESUMO
OBJECTIVE: Developmental epileptic encephalopathies (DEEs) are genetically heterogeneous severe childhood-onset epilepsies with developmental delay or cognitive deficits. In this study, we explored the pathogenic mechanisms of DEE-associated de novo mutations in the CACNA1A gene. METHODS: We studied the functional impact of four de novo DEE-associated CACNA1A mutations, including the previously described p.A713T variant and three novel variants (p.V1396M, p.G230V, and p.I1357S). Mutant cDNAs were expressed in HEK293 cells, and whole-cell voltage-clamp recordings were conducted to test the impacts on CaV 2.1 channel function. Channel localization and structure were assessed with immunofluorescence microscopy and three-dimensional (3D) modeling. RESULTS: We find that the G230V and I1357S mutations result in loss-of-function effects with reduced whole-cell current densities and decreased channel expression at the cell membrane. By contrast, the A713T and V1396M variants resulted in gain-of-function effects with increased whole-cell currents and facilitated current activation (hyperpolarized shift). The A713T variant also resulted in slower current decay. 3D modeling predicts conformational changes favoring channel opening for A713T and V1396M. SIGNIFICANCE: Our findings suggest that both gain-of-function and loss-of-function CACNA1A mutations are associated with similarly severe DEEs and that functional validation is required to clarify the underlying molecular mechanisms and to guide therapies.
Assuntos
Encefalopatias/genética , Canais de Cálcio/genética , Mutação com Ganho de Função , Síndrome de Lennox-Gastaut/genética , Mutação com Perda de Função , Espasmos Infantis/genética , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Técnicas de Patch-Clamp , FenótipoRESUMO
Stroke-like episodes (SLE) occur in phosphomannomutase deficiency (PMM2-CDG), and may complicate the course of channelopathies related to Familial Hemiplegic Migraine (FHM) caused by mutations in CACNA1A (encoding CaV2.1 channel). The underlying pathomechanisms are unknown. We analyze clinical variables to detect risk factors for SLE in a series of 43 PMM2-CDG patients. We explore the hypothesis of abnormal CaV2.1 function due to aberrant N-glycosylation as a potential novel pathomechanism of SLE and ataxia in PMM2-CDG by using whole-cell patch-clamp, N-glycosylation blockade and mutagenesis. Nine SLE were identified. Neuroimages showed no signs of stroke. Comparison of characteristics between SLE positive versus negative patients' group showed no differences. Acute and chronic phenotypes of patients with PMM2-CDG or CACNA1A channelopathies show similarities. Hypoglycosylation of both CaV2.1 subunits (α1A and α2α) induced gain-of-function effects on channel gating that mirrored those reported for pathogenic CACNA1A mutations linked to FHM and ataxia. Unoccupied N-glycosylation site N283 at α1A contributes to a gain-of-function by lessening CaV2.1 inactivation. Hypoglycosylation of the α2δ subunit also participates in the gain-of-function effect by promoting voltage-dependent opening of the CaV2.1 channel. CaV2.1 hypoglycosylation may cause ataxia and SLEs in PMM2-CDG patients. Aberrant CaV2.1 N-glycosylation as a novel pathomechanism in PMM2-CDG opens new therapeutic possibilities.
Assuntos
Doenças Cerebelares/complicações , Canalopatias/complicações , Fosfotransferases (Fosfomutases)/deficiência , Acidente Vascular Cerebral/complicações , Adolescente , Sequência de Aminoácidos , Canais de Cálcio/genética , Doenças Cerebelares/diagnóstico por imagem , Canalopatias/diagnóstico por imagem , Criança , Pré-Escolar , Eletroencefalografia , Feminino , Glicosilação , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Imageamento por Ressonância Magnética , Masculino , Mutação/genética , Fosfotransferases (Fosfomutases)/química , Fosfotransferases (Fosfomutases)/metabolismo , Acidente Vascular Cerebral/diagnóstico por imagem , Tunicamicina/farmacologiaRESUMO
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.
Assuntos
Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/ultraestrutura , Membrana Celular/química , Bicamadas Lipídicas/química , Células de Purkinje/química , ômega-Agatoxina IVA/química , Animais , Sítios de Ligação , Conformação Molecular , Ligação Proteica , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Action potentials are usually considered as the smallest unit of neuronal information conveyed by presynaptic neurons to their postsynaptic target. Thus, neuronal signaling in brain circuits is all-or-none or digital. However, recent studies indicate that subthreshold analog variation in presynaptic membrane potential modulates spike-evoked transmission. The informational content of each presynaptic action potential is therefore greater than initially expected. This property constitutes a form of fast activity-dependent modulation of functional coupling. Therefore, it could have important consequences on information processing in neural networks in parallel with more classical forms of presynaptic short-term facilitation based on repetitive stimulation, modulation of presynaptic calcium or modifications of the release machinery. We discuss here how analog voltage shift in the presynaptic neuron may regulate spike-evoked release of neurotransmitter through the modulation of voltage-gated calcium and potassium channels in the axon and presynaptic terminal. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio/metabolismo , Neurotransmissores/metabolismo , Canais de Potássio/metabolismo , Transmissão Sináptica/fisiologia , Animais , HumanosRESUMO
The ß subunits of voltage-gated calcium channels regulate surface expression and gating of CaV1 and CaV2 α1 subunits and thus contribute to neuronal excitability, neurotransmitter release, and calcium-induced gene regulation. In addition, certain ß subunits are targeted into the nucleus, where they interact directly with the epigenetic machinery. Whereas their involvement in this multitude of functions is reflected by a great molecular heterogeneity of ß isoforms derived from four genes and abundant alternative splicing, little is known about the roles of individual ß variants in specific neuronal functions. In the present study, an alternatively spliced ß4 subunit lacking the variable N terminus (ß4e) is identified. It is highly expressed in mouse cerebellum and cultured cerebellar granule cells (CGCs) and modulates P/Q-type calcium currents in tsA201 cells and CaV2.1 surface expression in neurons. Compared with the other two known full-length ß4 variants (ß4a and ß4b), ß4e is most abundantly expressed in the distal axon, but lacks nuclear-targeting properties. To determine the importance of nuclear targeting of ß4 subunits for transcriptional regulation, we performed whole-genome expression profiling of CGCs from lethargic (ß4-null) mice individually reconstituted with ß4a, ß4b, and ß4e. Notably, the number of genes regulated by each ß4 splice variant correlated with the rank order of their nuclear-targeting properties (ß4b > ß4a > ß4e). Together, these findings support isoform-specific functions of ß4 splice variants in neurons, with ß4b playing a dual role in channel modulation and gene regulation, whereas the newly detected ß4e variant serves exclusively in calcium-channel-dependent functions.
Assuntos
Canais de Cálcio/genética , Expressão Gênica/genética , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Canais de Cálcio/metabolismo , Feminino , Hipocampo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Patch-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Various CACNA1A missense mutations cause familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of migraine with aura. FHM1 mutation R192Q is associated with pure hemiplegic migraine, whereas the S218L mutation causes hemiplegic migraine, cerebellar ataxia, seizures, and mild head trauma-induced brain edema. Transgenic knock-in (KI) migraine mouse models were generated that carried either the FHM1 R192Q or the S218L mutation and were shown to exhibit increased CaV2.1 channel activity. Here we investigated their cerebellar and caudal cortical transcriptome. METHODS: Caudal cortical and cerebellar RNA expression profiles from mutant and wild-type mice were studied using microarrays. Respective brain regions were selected based on their relevance to migraine aura and ataxia. Relevant expression changes were further investigated at RNA and protein level by quantitative polymerase chain reaction (qPCR) and/or immunohistochemistry, respectively. RESULTS: Expression differences in the cerebellum were most pronounced in S218L mice. Particularly, tyrosine hydroxylase, a marker of delayed cerebellar maturation, appeared strongly upregulated in S218L cerebella. In contrast, only minimal expression differences were observed in the caudal cortex of either mutant mice strain. CONCLUSION: Despite pronounced consequences of migraine gene mutations at the neurobiological level, changes in cortical RNA expression in FHM1 migraine mice compared to wild-type are modest. In contrast, pronounced RNA expression changes are seen in the cerebellum of S218L mice and may explain their cerebellar ataxia phenotype.
Assuntos
Encéfalo/fisiopatologia , Canais de Cálcio Tipo N/genética , Ataxia Cerebelar/genética , Cerebelo/fisiopatologia , Córtex Cerebral/fisiopatologia , Transtornos de Enxaqueca/genética , Proteínas do Tecido Nervoso/genética , RNA/metabolismo , Animais , Ataxia Cerebelar/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transtornos de Enxaqueca/metabolismo , Mutação , RNA/genética , Distribuição Tecidual , Transcriptoma/genéticaRESUMO
Introduction: The emergent coherent population activity from thousands of stochastic neurons in the brain is believed to constitute a key neuronal mechanism for salient processing of external stimuli and its link to internal states like attention and perception. In the sensory cortex, functional cell assemblies are formed by recurrent excitation and inhibitory influences. The stochastic dynamics of each cell involved is largely orchestrated by presynaptic CAV2.1 voltage-gated calcium channels (VGCCs). Cav2.1 VGCCs initiate the release of neurotransmitters from the presynaptic compartment and are therefore able to add variability into synaptic transmission which can be partly explained by their mobile organization around docked vesicles. Methods: To investigate the relevance of Cav2.1 channel surface motility for the input processing in the primary auditory cortex (A1) in vivo, we make use of a new optogenetic system which allows for acute, reversable cross-linking Cav2.1 VGCCs via a photo-cross-linkable cryptochrome mutant, CRY2olig. In order to map neuronal activity across all cortical layers of the A1, we performed laminar current-source density (CSD) recordings with varying auditory stimulus sets in transgenic mice with a citrine tag on the N-terminus of the VGCCs. Results: Clustering VGCCs suppresses overall sensory-evoked population activity, particularly when stimuli lead to a highly synchronized distribution of synaptic inputs. Discussion: Our findings reveal the importance of membrane dynamics of presynaptic calcium channels for sensory encoding by dynamically adjusting network activity across a wide range of synaptic input strength.
RESUMO
Cardiovascular adjustments to exercise are partially mediated by group III/IV (small to medium) muscle afferents comprising the exercise pressor reflex (EPR). However, this reflex can be inappropriately activated in disease states (e.g., peripheral vascular disease), leading to increased risk of myocardial infarction. Here we investigate the voltage-dependent calcium (CaV) channels expressed in small to medium muscle afferent neurons as a first step toward determining their potential role in controlling the EPR. Using specific blockers and 5 mM Ba(2+) as the charge carrier, we found the major calcium channel types to be CaV2.2 (N-type) > CaV2.1 (P/Q-type) > CaV1.2 (L-type). Surprisingly, the CaV2.3 channel (R-type) blocker SNX482 was without effect. However, R-type currents are more prominent when recorded in Ca(2+) (Liang and Elmslie 2001). We reexamined the channel types using 10 mM Ca(2+) as the charge carrier, but results were similar to those in Ba(2+). SNX482 was without effect even though â¼27% of the current was blocker insensitive. Using multiple methods, we demonstrate that CaV2.3 channels are functionally expressed in muscle afferent neurons. Finally, ATP is an important modulator of the EPR, and we examined the effect on CaV currents. ATP reduced CaV current primarily via G protein ßγ-mediated inhibition of CaV2.2 channels. We conclude that small to medium muscle afferent neurons primarily express CaV2.2 > CaV2.1 ≥ CaV2.3 > CaV1.2 channels. As with chronic pain, CaV2.2 channel blockers may be useful in controlling inappropriate activation of the EPR.
Assuntos
Canais de Cálcio/metabolismo , Músculo Esquelético/inervação , Neurônios Aferentes/fisiologia , Potenciais de Ação , Trifosfato de Adenosina/farmacologia , Animais , Bário/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/classificação , Canais de Cálcio/genética , Linhagem Celular Tumoral , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Humanos , Masculino , Músculo Esquelético/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/metabolismo , Ratos , Ratos Sprague-Dawley , ReflexoRESUMO
Cav2.1 channels are expressed throughout the brain and are the predominant Ca2+ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.