RESUMO
Absolute transition frequencies of the 2s 2S{1/2}â2p2P{1/2,3/2} transitions in Be^{+} were measured for the isotopes ^{7,9-12}Be. The fine structure splitting of the 2p state and its isotope dependence are extracted and compared to results of ab initio calculations using explicitly correlated basis functions, including relativistic and quantum electrodynamics effects at the order of mα(6) and mα(7) â¢ln α. Accuracy has been improved in both the theory and experiment by 2 orders of magnitude, and good agreement is observed. This represents one of the most accurate tests of quantum electrodynamics for many-electron systems, being insensitive to nuclear uncertainties.
RESUMO
We present the concluding result from an Ives-Stilwell-type time dilation experiment using 7Li+ ions confined at a velocity of ß=v/c=0.338 in the storage ring ESR at Darmstadt. A Λ-type three-level system within the hyperfine structure of the 7Li+3S1 â3P2 line is driven by two laser beams aligned parallel and antiparallel relative to the ion beam. The lasers' Doppler shifted frequencies required for resonance are measured with an accuracy of <4×10(-9) using optical-optical double resonance spectroscopy. This allows us to verify the special relativity relation between the time dilation factor γ and the velocity ß, γâ1-ß2=1 to within ±2.3×10(-9) at this velocity. The result, which is singled out by a high boost velocity ß, is also interpreted within Lorentz invariance violating test theories.
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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
Chronic wounds associated with vascular disease, diabetes mellitus, or aging are leading causes of morbidity in western countries and represent an unresolved clinical problem. The development of innovative strategies to promote tissue repair is therefore an important task that requires a more thorough analysis of the underlying molecular pathophysiology. We propose that the understanding of the complex biological events that control tissue repair or its failure largely benefits from a broad analytical approach as provided by novel proteomic methodologies. Here we present the first comparative proteome analysis of wound exudates obtained from normal healing or nonhealing (venous leg ulcer) human skin wounds. A total of 149 proteins were identified with high confidence. A minority of proteins was exclusively present in exudate of the healing wound (23 proteins) or the nonhealing wound (26 proteins). Of particular interest was the differential distribution of specific proteins among the two different healing phenotypes. Whereas in the exudate obtained from the healing wound mediators characteristic for tissue formation were abundantly present, in the exudate obtained from the nonhealing wound numerous mediators characteristic for a persistent inflammatory and tissue destructive response were identified. Furthermore, the study also revealed interesting results regarding the identification of new proteins with yet unknown functions in skin repair. This analysis therefore represents an important basis for the search for potential biomarkers, which give rise to a better understanding and monitoring of disease progression in chronic wounds.
Assuntos
Exsudatos e Transudatos/química , Úlcera da Perna/metabolismo , Proteômica/métodos , Cicatrização/fisiologia , Idoso , Anexinas/química , Anexinas/metabolismo , Biomarcadores/química , Biomarcadores/metabolismo , Calgranulina B/química , Calgranulina B/metabolismo , Doença Crônica , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Humanos , Imuno-Histoquímica , Inflamação , Lactoferrina/química , Lactoferrina/metabolismo , Pessoa de Meia-Idade , Proteoma/química , Proteoma/metabolismo , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/farmacologia , Western Blotting , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Bovinos , Linhagem Celular , Guanidinas/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Lactose/farmacologia , Espectrometria de Massas , Modelos Biológicos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Perfusão , Fosforilação , Testes de Precipitina , Proteína Quinase C-alfa/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Retina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
There is growing evidence that Ca(v)2.3 (alpha1E, E-type) transcripts may encode the ion-conducting subunit of a subclass of R-type Ca(2+) channels, a heterogeneous group of channels by definition resistant to blockers of L-, N-, and P/Q-type Ca(2+) channels. To understand whether splice variation of Ca(v)2.3 contributes to the divergence of R-type channels, individual variants of Ca(v)2.3 were constructed and expressed in HEK-293 cells. With Ba(2+) as charge carrier, the tested biophysical properties were similar. In Ca(2+), the inactivation time course was slower and the recovery from short-term inactivation was faster; however, this occurred only in variants containing a 19-amino-acid-long insertion, which is typical for neuronal Ca(v)2.3 Ca(2+) channel subunits. This different Ca(2+) sensitivity is not responsible for the major differences between various R-type channels, and future studies might clarify its importance for in vivo synaptic or dendritic integration and the reasons for its loss in endocrine Ca(v)2.3 splice variants.
Assuntos
Processamento Alternativo/fisiologia , Canais de Cálcio/fisiologia , Proteínas de Transporte de Cátions , Fragmentos de Peptídeos/fisiologia , Potenciais de Ação/genética , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Canais de Cálcio/biossíntese , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio Tipo R , Linhagem Celular , Citosol/química , Citosol/fisiologia , Humanos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína/genética , Sintaxina 1RESUMO
There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Bário/farmacologia , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Linhagem Celular , Interações Medicamentosas , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Rim , Maleimidas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Fatores de Tempo , TransfecçãoRESUMO
A voltage-gated calcium channel containing Cav2.3e (alpha1Ee) as the ion conducting pore has recently been detected in rat heart. Functional evidence for this Ca2+ channel to be involved in the regulation of heart beating, besides L- and T-type channels, was derived from murine embryos where the gene for Cav1.2 had been ablated. The remaining "L-type like" current component was not related to recombinant splice variants of Cav1.3 containing channels. As recombinant Cav2.3 channels from rat were reported to be weakly dihydropyridine sensitive, the spontaneous activity of the prenatal hearts from Cav2.3(-|-) mice was compared to that of Cav2.3(+|+) control animals to investigate if Cav2.3 could represent such a L-type like Ca(2+) channel. The spontaneous activity of murine embryonic hearts was recorded by using a multielectrode array. Between day 9.5 p.c. to 12.5 p.c., the beating frequency of isolated embryonic hearts from Cav2.3-deficient mice did not differ significantly from control mice but the coefficient of variation within individual episodes was more than four-fold increased in Cav2.3-deficient mice indicating arrhythmia. In isolated hearts from wild type mice, arrhythmia was induced by superfusion with a solution containing 200 nM SNX-482, a blocker of some R-type voltage gated Ca2+ channels, suggesting that R-type channels containing the splice variant Cav2.3e as ion conducting pore stabilize a more regular heart beat in prenatal mice.