RESUMO
Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.
Assuntos
Processamento Alternativo , Canais de Cálcio Tipo R/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Calmodulina/química , Calmodulina/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Células HEK293 , Humanos , Imunoprecipitação , Dados de Sequência MolecularRESUMO
BACKGROUND: Voltage gated calcium channels (VGCCs) regulate cellular activity in response to membrane depolarization by altering calcium homeostasis. Because calcium is the most versatile second messenger, regulation of the amount of VGCCs at the plasma membrane is highly critical for several essential cellular processes. Among the different types of VGCCs, the Ca(v)2.3 calcium channel and its regulation mechanisms are least understood due to Ca(v)2.3's resistance to most pharmacological agents. METHODS: In order to study regulation and surface expression of Ca(v)2.3, a yeast two hybrid (Y2H) screen with the II-III loop of human Ca(v)2.3 as bait, was performed. APLP1, a member of the APP gene family and Rab5A, an endocytotic catalyst were identified as putative interaction partners. The interaction were confirmed by immunoprecipitation. To study the functional importance of the interaction, patch-clamp recordings in Ca(v)2.3 stably transfected HEK-293 cells (2C6) and surface biotin endocytosis assays were performed. RESULTS: We are able to show that the II-III loop of the Ca(v)2.3 calcium channel binds APLP1 and that this binding promotes internalization of the channel. In addition, Rab5A also binds to the same loop of the channel and exerts an inhibitory effect on APLP1 mediated channel internalization. CONCLUSIONS: This study identifies a regulation mechanism of Ca(v)2.3's surface expression, which implicates APLP1 as a regulator of calcium homeostasis. Thus APLP1 may play a crucial role in neuropathological mechanisms, which involve modulation of surface expression of voltage-gated Ca(2+) channels.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Canais de Cálcio Tipo R/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Precursor de Proteína beta-Amiloide/genética , Biotina/genética , Biotina/metabolismo , Canais de Cálcio Tipo R/química , Proteínas de Transporte de Cátions/química , Endocitose , Células HEK293 , Humanos , Imunoprecipitação , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Transfecção , Proteínas rab5 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Calcium channels are essential in coupling action potential to signal transduction in cells. There are several types of calcium channels, which can be pharmacologically classified as L-, N-, P/Q-, R- and T-type. But molecular basis of R-type channels is less clearly understood compared the other channel types. Therefore the current study aims at understanding the molecular functions of R-type calcium channels by identifying interaction partners of the channel. METHODS: In order to do so, a yeast two hybrid (Y2H) screen, with carboxy terminus of α1 subunit of the channel, as the bait, was performed. G1 subunit of v-ATPase was identified as a putative interaction partner of human Ca(v)2.3 by using the Y2H screening. The interaction was confirmed by immunoprecipitation. To study the functional importance of the interaction, bafilomycin A(1), a potent and specific inhibitor of v-ATPase was used in patch-clamp recordings in Ca(v)2.3 stably-transfected HEK-293 cells (2C6) as well as in electroretinography of the isolated bovine retina expressing R-type Ca(2+) channels. RESULTS: G1 subunit of v-ATPase interacts with C-terminal tail of Ca(v)2.3 and bafilomycin A(1) reduces Ca(v)2.3 mediated calcium currents. Additionally peak I(Ca) is inhibited in retinal signal transduction when recorded as ERG b-wave. CONCLUSIONS: The results suggest that v-ATPase interacts physically and also functionally with Ca(v)2.3. This is the first demonstration of association of Ca(v)2.3 C-terminus with a protein complex which is involved in transmembrane signalling.
Assuntos
Adenosina Trifosfatases/metabolismo , Canais de Cálcio Tipo R/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismo , Retina/fisiologia , Transdução de Sinais/fisiologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Animais , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Bovinos , Eletrorretinografia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Macrolídeos/farmacologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Plasmídeos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Screening for protein interaction partners of ion channels helps to elucidate signaling cascades to cellular targets and processes for a better understanding of the origin of diseases. Most important are the cytosolic segments of membrane-bound voltage- and ligand-gated ion channels or from ion channel regulators, which may connect to specific signaling complexes. So far, not much is known about those interactions. Molecular chaperones are proteins, which support the biosynthesis of proteins during maturation without being part of the final protein complex or which support the degradation of targeted proteins within the cellular protein quality control.
Assuntos
Proteínas de Choque Térmico/metabolismo , Canais Iônicos/metabolismo , Sequência de Aminoácidos , Animais , Descoberta de Drogas , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Alinhamento de Sequência , Transdução de SinaisRESUMO
Voltage-gated calcium channels are key components in the etiology and pathogenesis of epilepsies. Former studies mainly focused on P/Q-type Ca(v)2.1 and T-type Ca(v)3.2 Ca(2+) channels involved in absence epileptogenesis, but recent findings also point to an intriguing role of the Ca(v)2.3 E/R-type Ca(2+) channel in ictogenesis and seizure propagation. Based on the observation that Ca(v)2.3 is thought to induce plateau potentials in CA1 pyramidal cells, which can trigger epileptiform activity, our recent investigation revealed reduced PTZ-seizure susceptibility and altered seizure architecture in Ca(v)2.3(-/-) mice compared with controls. In the present study we tested hippocampal seizure susceptibility in Ca(v)2.3-deficient mice using surface and deep intrahippocampal telemetric EEG recordings as well as phenotypic seizure video analysis. Administration of kainic acid (30 mg/kg ip) revealed clear alteration in behavioral seizure architecture and dramatic resistance to limbic seizures in Ca(v)2.3(-/-) mice compared with controls, whereas no difference in hippocampal EEG seizure activity between both genotypes could be detected at this suprathreshold dosage. The same tendency was observed for NMDA seizure susceptibility (150 mg/kg ip) approaching the level of significance. In addition, histochemical analysis within the hippocampus revealed that excitotoxic effects after kainic acid administration are absent in Ca(v)2.3(-/-) mice, whereas Ca(v)2.3(+/+) animals exhibited clear and typical signs of excitotoxic cell death. These findings clearly indicate that the Ca(v)2.3 voltage-gated calcium channel plays a crucial role in both hippocampal ictogenesis and seizure generalization and is of central importance in neuronal degeneration after excitotoxic events.
Assuntos
Canais de Cálcio Tipo R/deficiência , Proteínas de Transporte de Cátions/deficiência , Hipocampo/fisiopatologia , Neurônios/fisiologia , Convulsões/etiologia , Convulsões/patologia , Animais , Eletroencefalografia/métodos , Hipocampo/efeitos dos fármacos , Ácido Caínico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Metilaspartato , Neurônios/efeitos dos fármacos , Convulsões/fisiopatologiaRESUMO
The Ca(v)2.3 encoded Ca2+ channel is probably one of the least well-understood voltage-gated calcium channels in terms of physiology, pharmacology and clinical relevance. Here we provide a detailed insight into the functional involvement of Ca(v)2.3 in etiology and pathogenesis of both convulsive and non-convulsive seizures. In the CNS, Ca(v)2.3 containing E/R-type Ca2+ channels are involved in triggering epileptiform discharges by significantly contributing to plateau potentials and afterdepolarisations. Pharmacological analysis further revealed that various antiepileptic drugs specifically target Ca(v)2.3 VGCCs capable of blocking epileptiform burst activity. Whereas electroencephalographic recordings in Ca(v)2.3-/- mice did not reveal any ictal-like discharges, seizure susceptibility was dramatically reduced in Ca(v)2.3-/- animals compared to controls, further supporting the observation that Ca(v)2.3 is an important factor in triggering epileptiform activity in neuronal populations. Although some aspects of its relationship to epilepsy have been uncovered, further functional characterization of Ca(v)2.3 in etiology and pathogenesis of human epileptic syndromes as well as development of new antiepileptic drugs specifically targeting Ca(v)2.3 turns out to become indispensable.
Assuntos
Canais de Cálcio Tipo R/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Epilepsia/etiologia , Epilepsia/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/genética , HumanosRESUMO
PURPOSE: Recently the Ca(v)2.3 (E/R-type) voltage-gated calcium channel (VGCC) has turned out to be not only a potential target for different antiepileptic drugs (e.g., lamotrigine, topiramate) but also a crucial component in the pathogenesis of absence epilepsy, human juvenile myoclonic epilepsy (JME), and epileptiform activity in CA1 neurons. The aim of our study was to perform an electroencephalographic analysis, seizure-susceptibility testing, and histomorphologic characterization of Ca(v)2.3-/- mice to unravel the functional relevance of Ca(v)2.3 in ictogenesis. METHODS: Generalized and brain-specific Ca(v)2.3 knockout animals were analyzed for spontaneous epileptiform discharges by using both electrocorticographic and deep intracerebral recordings. In addition, convulsive seizure activity was induced by systemic administration of either 4-aminopyridine (4-AP; 10 mg/kg, i.p.) or pentylenetetrazol (PTZ; 80 mg/kg, s.c.) to reveal possible alterations in seizure susceptibility. Besides histomorphologic analysis, expression studies of other voltage-gated Ca2+ channels in Ca(v)2.3-/- brains were carried out by using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Both electrocorticographic and deep intrahippocampal recordings exhibited no spontaneous epileptiform discharges indicative of convulsive or nonconvulsive seizure activity during long-term observation. Gross histology and expression levels of other voltage-gated Ca2+ channels remained unchanged in various brain regions. Surprisingly, PTZ-induced seizure susceptibility was dramatically reduced in Ca(v)2.3-deficient mice, whereas 4-AP sensitivity remained unchanged. CONCLUSIONS: Ca(v)2.3 ablation results in seizure resistance, strongly supporting recent findings in CA1 neurons that Ca(v)2.3 triggers epileptiform activity in specialized neurons via plateau potentials and afterdepolarizations. We provide novel insight into the functional involvement of Ca(v)2.3 in ictogenesis and seizure susceptibility on the whole-animal level.
Assuntos
Canais de Cálcio/fisiologia , Convulsões/induzido quimicamente , Convulsões/genética , 4-Aminopiridina/farmacologia , Animais , Western Blotting , Canais de Cálcio/genética , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/fisiologia , Modelos Animais de Doenças , Eletrodos Implantados/estatística & dados numéricos , Eletroencefalografia/estatística & dados numéricos , Eletromiografia/estatística & dados numéricos , Predisposição Genética para Doença/genética , Hipocampo/metabolismo , Hipocampo/patologia , Ativação do Canal Iônico/genética , Ativação do Canal Iônico/fisiologia , Camundongos , Camundongos Knockout , Pentilenotetrazol/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Convulsões/patologiaRESUMO
Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.