Importance of voltage-dependent inactivation in N-type calcium channel regulation by G-proteins.: Channel inactivation in G-protein regulation

International audienceDirect regulation of N-type calcium channels by G-proteins is essential to control neuronal excitability and neurotransmitter release. Binding of the G(betagamma) dimer directly onto the channel is characterized by a marked current inhibition ("ON" effect), whereas the pore opening- and time-dependent dissociation of this complex from the channel produce a characteristic set of biophysical modifications ("OFF" effects). Although G-protein dissociation is linked to channel opening, the contribution of channel inactivation to G-protein regulation has been poorly studied. Here, the role of channel inactivation was assessed by examining time-dependent G-protein de-inhibition of Ca(v)2.2 channels in the presence of various inactivation-altering beta subunit constructs. G-protein activation was produced via mu-opioid receptor activation using the DAMGO agonist. Whereas the "ON" effect of G-protein regulation is independent of the type of beta subunit, the "OFF" effects were critically affected by channel inactivation. Channel inactivation acts as a synergistic factor to channel activation for the speed of G-protein dissociation. However, fast inactivating channels also reduce the temporal window of opportunity for G-protein dissociation, resulting in a reduced extent of current recovery, whereas slow inactivating channels undergo a far more complete recovery from inhibition. Taken together, these results provide novel insights on the role of channel inactivation in N-type channel regulation by G-proteins and contribute to the understanding of the physiological consequence of channel inactivation in the modulation of synaptic activity by G-protein coupled receptors

Two PEST-like motifs regulate Ca2+/calpain-mediated cleavage of the CaVbeta3 subunit and provide important determinants for neuronal Ca2+ channel activity.

International audienceAn increase in intracellular Ca2+ due to voltage-gated Ca2+ (CaV) channel opening represents an important trigger for a number of second-messenger-mediated effects ranging from neurotransmitter release to gene activation. Ca2+ entry occurs through the principal pore-forming protein but several ancillary subunits are known to more precisely tune ion influx. Among them, the CaVbeta subunits are perhaps the most important, given that they largely influence the biophysical and pharmacological properties of the channel. Notably, several functional features may be associated with specific structural regions of the CaVbeta subunits emphasizing the relevance of intramolecular domains in the physiology of these proteins. In the current report, we show that CaVbeta3 contains two PEST motifs and undergoes Ca2+ -dependent degradation which can be prevented by the specific calpain inhibitor calpeptin. Using mutant constructs lacking the PEST motifs, we present evidence that they are necessary for the cleavage of CaVbeta3 by calpain. Furthermore, the deletion of the PEST sequences did not affect the binding of CaVbeta3 to the ion-conducting CaV2.2 subunit and, when expressed in human embryonic kidney-293 cells, the PEST motif-deleted CaVbeta3 significantly increased whole-cell current density and retarded channel inactivation. Consistent with this observation, calpeptin treatment of human embryonic kidney-293 cells expressing wild-type CaVbeta3 resulted in an increase in current amplitude. Together, these findings suggest that calpain-mediated CaVbeta3 proteolysis may be an essential process for Ca2+ channel functional regulation

Les déterminants moléculaires et cellulaires de la mutation humaine R482X de la sous-unité Cavb4 impliqués dans l'épilepsie

High voltage-activated (HVA) calcium channels are hetero-multimeric complexesthat translate electrical signals into Ca2+ influx, a secondary messenger that mediatesessential neuronal processes such as neurotransmitter release and neuronal excitability.HVA calcium channels are composed of four subunits: Cavα1 subunit, the pore formingof the channel and the auxiliary subunits Cavβ, Cavα2δ and Cavγ. The Cavß subunit hasfocused much of the interest owing to its regulatory functions within the complex. Bymasking an endoplasmic retention signal on the Cavα1 subunit, Cavß subunit targets themature calcium channel to the plasma membrane and contributes to the increase innumber of functional calcium currents. In humans, pathologic mutations have beenidentified in CACNAB4 that produce epileptic phenotype. One of these mutations is apremature termination mutation (R482X). Mutations produced minor biophysical effectson calcium channels in spite of their preponderant pathological phenotypes, which tend toindicate that cellular functions other than channel regulation may be responsible for thepredominant neurological effects of the R482X mutation.My thesis focuses on the molecular and cellular determinants of the Cavb4 mutant(R482X) inducing the human neurological phenotype. Studies were realized inhippocampal neurons which are particularly implicated in epilepsy. A translocation ofendogenous Cavß4, from the cytoplasm to the nucleus is observed during neuronaldifferentiation and synaptogenesis. This translocation is conditioned by the nativestructural conformation of Cavß4 subunit; carboxy-terminal deletion inhibits the nuclearlocalization of the mutant. The 38 amino acids deletion disturbs the structure of Cavß4 byaltering the intramolecular interaction between the two conserved domains in Cavß4subunit. In hippocampal neurons, this structural distortion prevents the nuclearlocalization of the mutant Cavß4. In order to identify the impact of the nuclear localizationof the Cavß4 subunit on transcriptional regulation, a study on gene expression generatedby microarray is realized. Profiles of gene expression revealed a differential generegulation between wild-type and mutant Cavß4. Cavß4 subunit seems to have a repressiveaction on gene regulation; on the other hand, the lack of nuclear localization of themutant reverses this repressive impact. Among the specific transcripts differentiallyexpressed, some genes are primordial keys in neuronal activities, thus alterations in theregulation of these genes could be involved in neuronal diseases. Since no NLSconsensus sequence has been identified on Cavß4, two-hybrid assay was realized in orderto identify partners responsible of the Cavß4 nuclear targeting. A specific protein holdinga NLS sequence interacts specifically with Cavß4 subunit, but not with the mutant, and isable to target Cavß4 to the nucleus. The other protein which specifically interacts with theWT Cavß4 sequesters Cavß4 in the cytoplasm.Finally, the lack in nuclear targeting of the R482X mutant due to the structuraldistortion appears to alter the transcriptional gene regulation which is maybe implicatedin the epileptic phenotype of patients holding the mutation.Les canaux calciques neuronaux activés par la dépolarisation membranairecontrôlent diverses fonctions cellulaires telles que l'excitabilité neuronale et latransmission synaptique. Les canaux calciques sont formés d'une sous-unité principaleCav associée à 3 sous-unités régulatrices (b, a2d et g). La sous-unité auxiliaire Cavb joueun rôle crucial dans la régulation des propriétés biophysiques du canal et dans l'adressagemembranaire de la sous-unité Cav. Chez l'homme, un isoforme de cette sous-unité Cavb4est l'objet de mutations qui conduisent à des phénotypes épileptiques. L'une de cesmutations est une délétion d'une partie du domaine carboxy-terminal de Cavb4 (R482X).Les efforts effectués pour comprendre les mécanismes cellulaires du phénotypeépileptique des patients concernés n'ont pas encore aboutit à des explications probantes.Ainsi, le phénotype neurologique ne semble pas lié à une altération de l'activité du canal,mais plus vraisemblablement à des fonctions cellulaires inconnues de Cavb4.Ma thèse porte sur la caractérisation des déterminants moléculaires et cellulairesdu mutant humain R482X impliqué dans le phénotype neurologique des patients quiportent la mutation. Etant donné que l'épilepsie implique essentiellement les neurones dulobe temporal, je me suis particulièrement intéressée à l'étude des fonctions et de lalocalisation de Cavb4 dans les neurones d'hippocampe. Dans ces cellules, unetranslocation de la sous-unité Cavb4 du cytoplasme vers le noyau est notée au cours de ladifférenciation neuronale. Cette translocation est dépendante de l'intégrité structurale dela sous-unité Cavb4, elle est perdue dans le cas de la mutation R482X. La perte dufragment carboxy-terminal conduit à une altération de la structure de Cavb4 suite à larupture de l'interaction intramoléculaire entre les deux domaines conservés, au sein de laprotéine native. Dans les neurones d'hippocampe, cette déstructuration empêche lalocalisation nucléaire de la protéine mutante. Etant donné que la sous-unité Cavb4 nepossède aucun signal d'adressage nucléaire, la technique du double hybride a été réaliséeafin de déterminer les partenaires protéiques capables d'adresser la sous-unité Cavb4 versle noyau. Parmi les trois protéines criblées qui interagissent spécifiquement avec Cavb4 etpas avec le mutant, une est capable d'adresser Cavb4 dans le noyau alors que l'autre laretient dans le cytoplasme. Afin d'étudier l'effet de la localisation nucléaire de la sousunitéCavb4 sur la régulation génique, une étude transcriptomique a été réalisée. La sousunitéCavb4 montre un effet répressif sur l'expression génique. Cette répression estinversée dans le cas du mutant incapable de s'adresser vers le noyau des neurones. Parmices gènes, plusieurs sont des candidats potentiels pour expliquer l'altération d'activitésneuronaux impliquée dans le phénotype épileptique.Enfin, l'absence de l'adressage nucléaire de la sous-unité Cavb4 mutante (R482X)due à la déformation de sa structure native, altèrerait la régulation transcriptionnelle desgènes, qui serait à la base du phénotype épileptique des patients qui portent cettemutation

Les déterminants moléculaires et cellulaires de la mutation humaine R482X de la sous-unité Cavb4 impliqués dans l épilepsie

Les canaux calciques neuronaux activés par la dépolarisation membranaire contrôlent diverses fonctions cellulaires telles que l excitabilité neuronale et la transmission synaptique. Les canaux calciques sont formés d une sous-unité principale Cav associée à 3 sous-unités régulatrices (b, a2d et g). La sous-unité auxiliaire Cavb joue un rôle crucial dans la régulation des propriétés biophysiques du canal et dans l adressage membranaire de la sous-unité Cav. Chez l homme, un isoforme de cette sous-unité Cavb4 est l objet de mutations qui conduisent à des phénotypes épileptiques. L une de ces mutations est une délétion d une partie du domaine carboxy-terminal de Cavb4 (R482X). Les efforts effectués pour comprendre les mécanismes cellulaires du phénotype épileptique des patients concernés n ont pas encore aboutit à des explications probantes. Ainsi, le phénotype neurologique ne semble pas lié à une altération de l activité du canal, mais plus vraisemblablement à des fonctions cellulaires inconnues de Cavb4. Ma thèse porte sur la caractérisation des déterminants moléculaires et cellulaires du mutant humain R482X impliqué dans le phénotype neurologique des patients qui portent la mutation. Etant donné que l épilepsie implique essentiellement les neurones du lobe temporal, je me suis particulièrement intéressée à l étude des fonctions et de la localisation de Cavb4 dans les neurones d hippocampe. Dans ces cellules, une translocation de la sous-unité Cavb4 du cytoplasme vers le noyau est notée au cours de la différenciation neuronale. Cette translocation est dépendante de l intégrité structurale de la sous-unité Cavb4, elle est perdue dans le cas de la mutation R482X. La perte du fragment carboxy-terminal conduit à une altération de la structure de Cavb4 suite à la rupture de l interaction intramoléculaire entre les deux domaines conservés, au sein de la protéine native. Dans les neurones d hippocampe, cette déstructuration empêche la localisation nucléaire de la protéine mutante. Etant donné que la sous-unité Cavb4 ne possède aucun signal d adressage nucléaire, la technique du double hybride a été réalisée afin de déterminer les partenaires protéiques capables d adresser la sous-unité Cavb4 vers le noyau. Parmi les trois protéines criblées qui interagissent spécifiquement avec Cavb4 et pas avec le mutant, une est capable d adresser Cavb4 dans le noyau alors que l autre la retient dans le cytoplasme. Afin d étudier l effet de la localisation nucléaire de la sousunité Cavb4 sur la régulation génique, une étude transcriptomique a été réalisée. La sousunité Cavb4 montre un effet répressif sur l expression génique. Cette répression est inversée dans le cas du mutant incapable de s adresser vers le noyau des neurones. Parmi ces gènes, plusieurs sont des candidats potentiels pour expliquer l altération d activités neuronaux impliquée dans le phénotype épileptique. Enfin, l absence de l adressage nucléaire de la sous-unité Cavb4 mutante (R482X) due à la déformation de sa structure native, altèrerait la régulation transcriptionnelle des gènes, qui serait à la base du phénotype épileptique des patients qui portent cette mutationHigh voltage-activated (HVA) calcium channels are hetero-multimeric complexes that translate electrical signals into Ca2+ influx, a secondary messenger that mediates essential neuronal processes such as neurotransmitter release and neuronal excitability. HVA calcium channels are composed of four subunits: Cava1 subunit, the pore forming of the channel and the auxiliary subunits Cavb, Cava2 and Cavg. The Cavß subunit has focused much of the interest owing to its regulatory functions within the complex. By masking an endoplasmic retention signal on the Cava1 subunit, Cavß subunit targets the mature calcium channel to the plasma membrane and contributes to the increase in number of functional calcium currents. In humans, pathologic mutations have been identified in CACNAB4 that produce epileptic phenotype. One of these mutations is a premature termination mutation (R482X). Mutations produced minor biophysical effects on calcium channels in spite of their preponderant pathological phenotypes, which tend to indicate that cellular functions other than channel regulation may be responsible for the predominant neurological effects of the R482X mutation. My thesis focuses on the molecular and cellular determinants of the Cavb4 mutant (R482X) inducing the human neurological phenotype. Studies were realized in hippocampal neurons which are particularly implicated in epilepsy. A translocation of endogenous Cavß4, from the cytoplasm to the nucleus is observed during neuronal differentiation and synaptogenesis. This translocation is conditioned by the native structural conformation of Cavß4 subunit; carboxy-terminal deletion inhibits the nuclear localization of the mutant. The 38 amino acids deletion disturbs the structure of Cavß4 by altering the intramolecular interaction between the two conserved domains in Cavß4 subunit. In hippocampal neurons, this structural distortion prevents the nuclear localization of the mutant Cavß4. In order to identify the impact of the nuclear localization of the Cavß4 subunit on transcriptional regulation, a study on gene expression generated by microarray is realized. Profiles of gene expression revealed a differential gene regulation between wild-type and mutant Cavß4. Cavß4 subunit seems to have a repressive action on gene regulation; on the other hand, the lack of nuclear localization of the mutant reverses this repressive impact. Among the specific transcripts differentially expressed, some genes are primordial keys in neuronal activities, thus alterations in the regulation of these genes could be involved in neuronal diseases. Since no NLS consensus sequence has been identified on Cavß4, two-hybrid assay was realized in order to identify partners responsible of the Cavß4 nuclear targeting. A specific protein holding a NLS sequence interacts specifically with Cavß4 subunit, but not with the mutant, and is able to target Cavß4 to the nucleus. The other protein which specifically interacts with the WT Cavß4 sequesters Cavß4 in the cytoplasm. Finally, the lack in nuclear targeting of the R482X mutant due to the structural distortion appears to alter the transcriptional gene regulation which is maybe implicated in the epileptic phenotype of patients holding the mutationGRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Towards filling the gaps around recurrent events in high dimensional framework: a systematic literature review and application

International audienceIndividuals may experience repeated events over time. However, there is no consensus about learning approaches to use in a high-dimensional framework for survival data (when the number of variables exceeds the number of individuals, i.e. p > n). The aim of this study was to identify learning algorithms for analyzing/predicting recurrent events, and to compare them to standard statistical models on simulated data. A systematic literature review was conducted to provide state-of-the-art methodology. Data were then simulated according to the number of variables, the proportion of active variables, and the number of events. The performance of the models was assessed using Harrell’s concordance index, Kim’s C-index, and error rate for active variables. Seven publications were identified, of which four were methodological studies, one an application paper and two were reviews. On simulated data, the standard models failed when p > n. Penalized Andersen–Gill and frailty models outperformed, whereas RankDeepSurv gave poorer performances. With no current guidelines on a specific approach to use, this study deepens understanding of the mechanisms and limits of investigated methods in this context

Clinical Relevance of Cluster Analysis in Phenotyping Allergic Rhinitis in a Real-Life Study

International audienc