RESUMEN
CaV3.3 is the third member of the low-voltage-activated calcium channel family and the last to be recognized as disease gene. Previously, CACNA1I, the gene encoding CaV3.3, had been described as schizophrenia risk gene. More recently, de novo missense mutations in CACNA1I were identified in patients with variable degrees of neurodevelopmental disease with and without epilepsy. Their functional characterization indicated gain-of-function effects resulting in increased calcium load and hyperexcitability of neurons expressing CaV3.3. The amino acids mutated in the CaV3.3 disease variants are located in the vicinity of the channel's activation gate and thus are classified as gate-modifying channelopathy mutations. A persistent calcium leak during rest and prolonged calcium spikes due to increased voltage sensitivity of activation and slowed kinetics of channel inactivation, respectively, may be causal for the neurodevelopmental defects. The prominent expression of CaV3.3 in thalamic reticular nucleus neurons and its essential role in generating the rhythmic thalamocortical network activity are consistent with a role of the mutated channels in the etiology of epileptic seizures and thus suggest T-type channel blockers as a viable treatment option.
Asunto(s)
Canales de Calcio Tipo T , Canalopatías , Humanos , Canalopatías/genética , Calcio/metabolismo , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/química , Canales de Calcio Tipo T/metabolismo , MutaciónRESUMEN
P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and ß4 subunits. ß4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat ß4b cDNA. Heterologously expressed wild-type ß4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the ß4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, ß4b and ß4b-L125P augmented the calcium current amplitudes, however, ß4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of ß4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype ß4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of ß4b in cultured myotubes and hippocampal neurons. While binding of ß4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between ß4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of ß4b.
Asunto(s)
Canales de Calcio/genética , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Subunidades de Proteína/genética , Animales , Calcio/metabolismo , Canales de Calcio Tipo N/genética , Células Cultivadas , Femenino , Regulación de la Expresión Génica/genética , Células HEK293 , Hipocampo/fisiología , Homocigoto , Humanos , Masculino , Ratones Endogámicos BALB C , Neuronas/metabolismo , Terminales Presinápticos/fisiología , Isoformas de Proteínas/genética , Ratas , Transmisión Sináptica/genéticaRESUMEN
The skeletal muscle CaV 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium-dependent inactivation (CDI) of L-type calcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV 1.3 currents. When coexpressed in CaV 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with CaV 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports CaV 1.1 functional expression and EC coupling, although at a reduced extent compared to wild-type STAC3. Importantly, STAC3-ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV 1.1 currents, suggesting that STAC3 determines the slow CaV 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3-ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage-dependent inactivation (VDI) of CaV 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.
Asunto(s)
Calcio , Acoplamiento Excitación-Contracción , Calcio/metabolismo , Acoplamiento Excitación-Contracción/fisiología , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Señalización del Calcio/fisiología , CinéticaRESUMEN
T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Activación del Canal Iónico/genética , Trastornos del Neurodesarrollo/genética , Adulto , Animales , Encéfalo/metabolismo , Encéfalo/patología , Niño , Simulación por Computador , Femenino , Mutación con Ganancia de Función , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Ratones , Persona de Mediana Edad , Modelos Moleculares , Modelos Neurológicos , Mutación Missense , Neuronas/metabolismo , Linaje , Conformación ProteicaRESUMEN
The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.
Asunto(s)
Canales de Calcio Tipo L , Activación del Canal Iónico , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Cationes , Técnicas de Placa-ClampRESUMEN
The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1-/-) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Canales de Calcio Tipo L/genética , Calmodulina/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Moscas Domésticas , Humanos , Ratones , Fibras Musculares Esqueléticas/citología , Proteínas del Tejido Nervioso/genética , Técnicas de Placa-Clamp , Dominios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
CaV1.1 is specifically expressed in skeletal muscle where it functions as voltage sensor of skeletal muscle excitation-contraction (EC) coupling independently of its functions as L-type calcium channel. Consequently, all known CaV1.1-related diseases are muscle diseases and the molecular and cellular disease mechanisms relate to the dual functions of CaV1.1 in this tissue. To date, four types of muscle diseases are known that can be linked to mutations in the CACNA1S gene or to splicing defects. These are hypo- and normokalemic periodic paralysis, malignant hyperthermia susceptibility, CaV1.1-related myopathies, and myotonic dystrophy type 1. In addition, the CaV1.1 function in EC coupling is perturbed in Native American myopathy, arising from mutations in the CaV1.1-associated protein STAC3. Here, we first address general considerations concerning the possible roles of CaV1.1 in disease and then discuss the state of the art regarding the pathophysiology of the CaV1.1-related skeletal muscle diseases with an emphasis on molecular disease mechanisms.
Asunto(s)
Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Canalopatías/genética , Canalopatías/metabolismo , Músculo Esquelético/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Canalopatías/patología , Humanos , Músculo Esquelético/patología , Mutación/genéticaRESUMEN
Excitation-contraction (EC) coupling in skeletal muscle requires functional and mechanical coupling between L-type voltage-gated calcium channels (CaV1.1) and the ryanodine receptor (RyR1). Recently, STAC3 was identified as an essential protein for EC coupling and is part of a group of three proteins that can bind and modulate L-type voltage-gated calcium channels. Here, we report crystal structures of tandem-SH3 domains of different STAC isoforms up to 1.2-Å resolution. These form a rigid interaction through a conserved interdomain interface. We identify the linker connecting transmembrane repeats II and III in two different CaV isoforms as a binding site for the SH3 domains and report a crystal structure of the complex with the STAC2 isoform. The interaction site includes the location for a disease variant in STAC3 that has been linked to Native American myopathy (NAM). Introducing the mutation does not cause misfolding of the SH3 domains, but abolishes the interaction. Disruption of the interaction via mutations in the II-III loop perturbs skeletal muscle EC coupling, but preserves the ability of STAC3 to slow down inactivation of CaV1.2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Canales de Calcio Tipo L/metabolismo , Animales , Sitios de Unión/fisiología , Calcio/metabolismo , Señalización del Calcio/fisiología , Fisura del Paladar/metabolismo , Cristalografía por Rayos X/métodos , Acoplamiento Excitación-Contracción/fisiología , Humanos , Hipertermia Maligna/metabolismo , Proteínas de la Membrana/metabolismo , Músculo Esquelético/metabolismo , Mutación/genética , Miotonía Congénita/metabolismo , Isoformas de Proteínas/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Xenopus laevis/metabolismoRESUMEN
Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio Tipo L/genética , Acoplamiento Excitación-Contracción/genética , Fibras Musculares de Contracción Rápida/citología , Fibras Musculares de Contracción Lenta/citología , Debilidad Muscular/genética , Músculo Esquelético/embriología , Empalme Alternativo/genética , Animales , Calcio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares de Contracción Rápida/fisiología , Fibras Musculares de Contracción Lenta/fisiología , Debilidad Muscular/metabolismo , Isoformas de Proteínas/genéticaRESUMEN
Excitation-contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine-rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.
Asunto(s)
Canales de Calcio Tipo L/genética , Enfermedades Musculares/genética , Proteínas del Tejido Nervioso/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Calcio/metabolismo , Señalización del Calcio/genética , Acoplamiento Excitación-Contracción/genética , Acoplamiento Excitación-Contracción/fisiología , Humanos , Ratones , Fibras Musculares Esqueléticas , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Enfermedades Musculares/fisiopatologíaRESUMEN
Voltage-gated calcium channels represent the sole mechanism converting electrical signals of excitable cells into cellular functions such as contraction, secretion and gene regulation. Specific voltage-sensing domains detect changes in membrane potential and control channel gating. Calcium ions entering through the channel function as second messengers regulating cell functions, with the exception of skeletal muscle, where CaV 1.1 essentially does not function as a channel but activates calcium release from intracellular stores. It has long been known that calcium currents are dispensable for skeletal muscle contraction. However, the questions as to how and why the channel function of CaV 1.1 is curtailed remained obscure until the recent discovery of a developmental CaV 1.1 splice variant with normal channel functions. This discovery provided new means to study the molecular mechanisms regulating the channel gating and led to the understanding that in skeletal muscle, calcium currents need to be restricted to allow proper regulation of fibre type specification and to prevent mitochondrial damage.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Músculo Esquelético/fisiología , Animales , Calcio/fisiología , Canales de Calcio Tipo L/química , Humanos , Enfermedades Musculares/fisiopatología , Conformación ProteicaRESUMEN
In skeletal muscle excitation-contraction coupling, a voltage-gated calcium channel directly activates opening of the calcium release channel (RyR1) in the sarcoplasmic reticulum that supplies the calcium signal triggering contraction. In addition, a retrograde signal from the RyR1 facilitates gating of the voltage-gated calcium channel. Recent studies of RyR1 mutants, including the article by Bannister et al. in this issue of the Biophysical Journal, advance our understanding of the signaling mechanism, although the physiological significance of retrograde coupling remains elusive.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Señalización del Calcio , Acoplamiento Excitación-Contracción , Humanos , Contracción Muscular , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Transducción de SeñalRESUMEN
L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function.
Asunto(s)
Canales de Calcio Tipo L/química , Canales de Calcio/química , Calcio/metabolismo , Secuencia de Aminoácidos , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Línea Celular Transformada , Membrana Celular/química , Membrana Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Expresión Génica , Humanos , Transporte Iónico , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Fosfolipasas de Tipo C/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
The auxiliary voltage-gated calcium channel subunit ß4 supports targeting of calcium channels to the cell membrane, modulates ionic currents and promotes synaptic release in the central nervous system. ß4 is abundant in cerebellum and its loss causes ataxia. However, the type of calcium channels and cerebellar functions affected by the loss of ß4 are currently unknown. We therefore studied the structure and function of Purkinje cells in acute cerebellar slices of the ß4 (-/-) ataxic (lethargic) mouse, finding that loss of ß4 affected Purkinje cell input, morphology and pacemaker activity. In adult lethargic cerebellum evoked postsynaptic currents from parallel fibres were depressed, while paired-pulse facilitation and spontaneous synaptic currents were unaffected. Because climbing fibre input was spared, the parallel fibre/climbing fibre input ratio was reduced. The dendritic arbor of adult lethargic Purkinje cells displayed fewer and shorter dendrites, but a normal spine density. Accordingly, the width of the molecular and granular layers was reduced. These defects recapitulate the impaired cerebellar maturation observed upon Cav 2.1 ataxic mutations. However, unlike Cav 2.1 mutations, lethargic Purkinje cells also displayed a striking decrease in pacemaker firing frequency, without loss of firing regularity. All these deficiencies appear in late development, indicating the importance of ß4 for the normal differentiation and function of mature Purkinje cells networks. The observed reduction of the parallel fibre input, the altered parallel fibre/climbing fibre ratio and the reduced Purkinje cell output can contribute to the severe motor impairment caused by the loss of the calcium channel ß4 subunit in lethargic mice.
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Potenciales de Acción , Ataxia/fisiopatología , Canales de Calcio/fisiología , Células de Purkinje/fisiología , Animales , Canales de Calcio/genética , Espinas Dendríticas , Femenino , Masculino , Ratones , Ratones Noqueados , Células de Purkinje/citología , Potenciales SinápticosRESUMEN
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
CaV1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult CaV1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant CaV1.1a, the embryonic CaV1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike CaV1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in CaV1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (CaV1.1-null) myotubes reconstituted with either CaV1.1a or CaV1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric CaV1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in CaV1.1a had abolished the calcium currents, substitution of the II-III loop in CaV1.1e did not significantly reduce current amplitudes. This indicates that CaV1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in CaV1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, CaV1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 µM for current inhibition by nifedipine, CaV1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant CaV1.1e in the skeletal muscles of myotonic dystrophy patients.
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Canales de Calcio Tipo L/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/genética , Línea Celular Tumoral , Acoplamiento Excitación-Contracción , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Nifedipino/farmacología , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
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.
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Canales de Calcio/genética , Expresión Génica/genética , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Canales de Calcio/metabolismo , Femenino , Hipocampo/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Placa-Clamp , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Voltage-gated calcium channels (VGCCs) represent the sole mechanism to convert membrane depolarization into cellular functions like secretion, contraction, or gene regulation. VGCCs consist of a pore-forming α(1) subunit and several auxiliary channel subunits. These subunits come in multiple isoforms and splice-variants giving rise to a stunning molecular diversity of possible subunit combinations. It is generally believed that specific auxiliary subunits differentially regulate the channels and thereby contribute to the great functional diversity of VGCCs. If auxiliary subunits can associate and dissociate from pre-existing channel complexes, this would allow dynamic regulation of channel properties. However, most auxiliary subunits modulate current properties very similarly, and proof that any cellular calcium channel function is indeed modulated by the physiological exchange of auxiliary subunits is still lacking. In this review we summarize available information supporting a differential modulation of calcium channel functions by exchange of auxiliary subunits, as well as experimental evidence in support of alternative functions of the auxiliary subunits. At the heart of the discussion is the concept that, in their native environment, VGCCs function in the context of macromolecular signaling complexes and that the auxiliary subunits help to orchestrate the diverse protein-protein interactions found in these calcium channel signalosomes. Thus, in addition to a putative differential modulation of current properties, differential subcellular targeting properties and differential protein-protein interactions of the auxiliary subunits may explain the need for their vast molecular diversity.
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Canales de Calcio/química , Calcio/metabolismo , Isoformas de Proteínas/química , Canales de Calcio/metabolismo , Humanos , Neuronas/química , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
Voltage-gated Ca(2+) channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary ß subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether ß subunits can reversibly interact with, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged α1 and ß subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca(2+) channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle ß1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle ß2a and ß4b isoforms dynamically interacted with both α1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions in the proximal I-II loop, known to change the orientation of ß relative to the α1 subunit, altered the specific dynamic properties of the ß subunits. In contrast, a single residue substitution in the α interaction pocket of ß1aM293A increased the FRAP rate threefold. Taken together, these findings indicate that in skeletal muscle triads the homologous ß1a subunit forms a stable complex, whereas the heterologous ß2a and ß4b subunits form dynamic complexes with the Ca(2+) channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the α1 subunits, but are intrinsic properties of the ß subunit isoforms.
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Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo N/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Proteínas Musculares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Subunidades de Proteína , RatasRESUMEN
The murine embryonic diaphragm is a primary model for studying myogenesis and neuro-muscular synaptogenesis, both representing processes regulated by spatially organized genetic programs of myonuclei located in distinct myodomains. However, a spatial gene expression pattern of embryonic mouse diaphragm has not been reported. Here, we provide spatially resolved gene expression data for horizontally sectioned embryonic mouse diaphragms at embryonic days E14.5 and E18.5. These data reveal gene signatures for specific muscle regions with distinct maturity and fiber type composition, as well as for a central neuromuscular junction (NMJ) and a peripheral myotendinous junction (MTJ) compartment. Comparing spatial expression patterns of wild-type mice with those of transgenic mice lacking either the skeletal muscle calcium channel CaV1.1 or ß-catenin, reveals curtailed muscle development and dysregulated expression of genes potentially involved in NMJ formation. Altogether, these datasets provide a powerful resource for further studies of muscle development and NMJ formation in the mouse.