Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels.

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The alpha 1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between alpha 1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the alpha 1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the alpha 1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

Determinants of the differential gating properties of Cav3.1 and Cav3.3 T-type channels: a role of domain IV?

We have investigated the channel structural determinants that underlie the difference in gating properties of Cav3.1 and Cav3.3 T-type channels, by creating a series of chimeric channel constructs in which the major transmembrane domains were swapped. The chimeras were then expressed in tsA-201 cells and subjected to whole cell patch clamp analysis. Our data reveal that domains I and IV are major determinants of the half-activation potential. Substitution of domain IV was the most important determinant of activation time constant and time constant for recovery from inactivation, with domains I and II mediating a smaller role. In contrast, the carboxy terminal region did not appear to be involved. Determinants of the time constant for inactivation could not be localized to a specific transmembrane domain, but the concomitant substitution of domains I+IV was able to partially confer the inactivation kinetics among the two wild type channels. Our data indicate that the domain IV region mediates an important role in T-type channel activation, whereas multiple channel structural determinants appear to control T-type channel inactivation.

Overexpression of T-type calcium channels in HEK-293 cells increases intracellular calcium without affecting cellular proliferation.

Increased expression of low voltage-activated, T-type Ca(2+) channels has been correlated with a variety of cellular events including cell proliferation and cell cycle kinetics. The recent cloning of three genes encoding T-type alpha(1) subunits, alpha(1G), alpha(1H) and alpha(1I), now allows direct assessment of their involvement in mediating cellular proliferation. By overexpressing the human alpha(1G) and alpha(1H) subunits in human embryonic kidney (HEK-293) cells, we describe here that, although T-type channels mediate increases in intracellular Ca(2+) concentrations, there is no significant change in bromodeoxyuridine incorporation and flow cytometric analysis. These results demonstrate that expressions of T-type Ca(2+) channels are not sufficient to modulate cellular proliferation of HEK-293 cells.

New substituted 1,4-benzoxazine derivatives with potential intracellular calcium activity.

Substituted 1,4-benzoxazines bearing an amino side chain at the 2-position were prepared and were found to have a moderate activity on intracellular calcium. Of the compounds studied it was found that those which possess a homoveratrylamino moiety exhibited superior potency. The chain length and the nature of the amine (4-fluorophenylpiperazine, 4-fluorobenzhydryloxyethylamine, N-substituted homoveratrylamine) is discussed. The 4-benzyl-3, 4-dihydro-2-[3-[[2-(3,4-dimethoxyphenyl)ethyl]amino]propyl]-2H-1, 4-benzoxazine (3c) is the most potent derivative of the series with a ratio of IC50 values against PE (phenylephrine) and K+ of 2.1. Under these test conditions a ratio near 1 indicates potential intracellular calcium activity while a ratio greater than 100 an action on extracellular calcium influx.

Design and synthesis of substituted compounds containing the 1, 4-benzodioxin subunit. New potential calcium antagonists.

New compounds possessing 1,4-benzodioxin or its saturated analogous heterocyclic system were synthesized and tested for calcium antagonist activity. Biological differences were seen between the different modifications applied. These compounds have been shown to be representative of a novel series of calcium channel antagonists.

Synthesis of various analog derivatives of ORG13514 as 5-HT1A ligands.

In connection with the development of new potential 5-HT1A ligands, multistep synthesis of N-substituted-3-aminomethyl-2,3-dihydro-1,4-dioxinol[2,3-b]pyridin e derivatives as ORG13514 analogs are described. Their biological activity as 5-HT1A type ligands is reported and compared with ORG13514 affinity and selectivity for 5-HT1A receptors.

[Molecular genetics of cardiovascular calcium channels]. / Génétique moléculaire des canaux calciques cardiovasculaires.

Cardiac and vascular myocytes exhibit L type calcium channel currents with slightly different properties. The structural bases of this concept of functional diversity of cardiovascular calcium channels are now known. Firstly, there are multiple isoforms of the alpha 1 pore subunit. In addition, the beta subunit should be presented as an endogenous regulator of the calcium channel. Like the alpha 1 subunit, there are many isoforms of this regulatory subunit. A series of recent experiments has changed our understanding of the mechanisms which govern the expression of a functional diversity of calcium channels in the cardiovascular cells. Several pharmacological sites, such as the dihydropyridine, phenylalkylamine and benzothiazepine receptors, have been identified. The recent developments in the field of molecular genetics of the calcium channels are many and open up new perspectives. Mutations within these channels could be the cause of certain cardiovascular genetic diseases.

[Molecular diversity of calcium channel activities by depolarization]. / Diversité moléculaire des canaux calciques activés par la dépolarisation.

Voltage-gated calcium channels are involved in a large variety of cellular functions such as excitation-contraction coupling, hormone secretion, firing and pacemaker activity, gene activation and proliferation. Cloning of complementary DNAs encoding for calcium channel subunits has challenged the study of the functional properties of calcium channels and has allowed analysis of the molecular basis of calcium channel diversity. Recently, pore-forming subunits of T-type calcium channels have been cloned. Recent data describing the genes encoding calcium channels, their molecular and pharmacological studies, as well as their linkage to human genetic diseases are reviewed in this article.

[Creutzfeldt-Jakob disease and psychiatry. Apropos of a case]. / Maladie de Creutzfeldt-Jakob et psychiatrie. A propos d'un cas.

Creutzfeldt-Jakob disease is relatively poorly documented in psychiatry. An observation will be presented concerning a 69 years old patient admitted for conduct disorder and prosecution delirium. In fact, the symptomatology associated dementia, transient confusion, parkinsonism, ataxia and abnormal movements. In the checkup the electroencephalogram gave us an important orientation for diagnosis. After a thoracic trauma the patient was admitted in an intensive care unit with a positive pressure ventilation. His general condition worsened until he died. Brain histic exploration and PRP gene analysis confirmed the diagnosis. Even if there is no efficient treatment, such a diagnosis has to be done in order to take prophylactic measures and conduct epidemiological surveys.

[Physiopathology of calcium channels: identification of calcium channelopathies]. / Physiopathologie des canaux calciques: identification des canalopathies calciques.

Since a few years, many mutations in genes encoding voltage-dependent ion channels have been identified. The related disorders are quoted as "channelopathies". These mutations are responsible for several skeletal muscle, brain, heart or kidney diseases. Abnormal calcium channels genes are responsible for hypokaleamic periodic paralysis (CACNA1S) as well as some forms of ataxia, cerebellar degeneration and migraine (CACNA1A). The preliminary studies of the recently discovered calcium channelopathies are undergoing. Both in vitro and in vivo studies of the diseased genes should help to the understanding of the related pathologies as well as to extend our knowledge of calcium channel function. In addition, autoantibodies against calcium channels are retrieved in some autoimmune diseases, such as Lambert-Eaton myasthenic syndrome (LEMS). Complementary studies are necessary to identify the precise implication of calcium channels in these auto-immune channelopathies.

[2 cases of anaphylactic-type reaction to alphadione (Alfatesine, Althesin, CT 13.41)]. / A propos de deux observations de réaction de type anaphylactique a l'alfadione (Alfatésine -Althésin -CT 13.41)

Two cases of anaphylactic-type reaction to alfadione were referred to our Department for immuno-allergic enquiry. From the clinical standpoint, the pictures obtained were rather similar, dominated by dramatic collapse of abrupt onset associated with an erythemato-oedematous cervico-facial rash, however with a favourable course under respiratory and circulatory emergency resuscitation. The immunological enquiry carried out in both cases did not enable one to attribute the responsibility for the accident to alfadione or to cremophor EL nor to be able to put it down to a direct pharmacological histamine-liberation, or to a specific histamine-liberation due to an antigen-antibody reaction.

Alternatively spliced alpha(1G) (Ca(V)3.1) intracellular loops promote specific T-type Ca(2+) channel gating properties.

At least three genes encode T-type calcium channel alpha(1) subunits, and identification of cDNA transcripts provided evidence that molecular diversity of these channels can be further enhanced by alternative splicing mechanisms, especially for the alpha(1G) subunit (Ca(V)3.1). Using whole-cell patch-clamp procedures, we have investigated the electrophysiological properties of five isoforms of the human alpha(1G) subunit that display a distinct III-IV linker, namely, alpha(1G-a), alpha(1G-b), and alpha(1G-bc), as well as a distinct II-III linker, namely, alpha(1G-ae), alpha(1G-be), as expressed in HEK-293 cells. We report that insertion e within the II-III linker specifically modulates inactivation, steady-state kinetics, and modestly recovery from inactivation, whereas alternative splicing within the III-IV linker affects preferentially kinetics and voltage dependence of activation, as well as deactivation and inactivation. By using voltage-clamp protocols mimicking neuronal activities, such as cerebellar train of action potentials and thalamic low-threshold spike, we describe that inactivation properties of alpha(1G-a) and alpha(1G-ae) isoforms can support channel behaviors reminiscent to those described in native neurons. Altogether, these data demonstrate that expression of distinct variants for the T-type alpha(1G) subunit can account for specific low-voltage-activated currents observed in neuronal tissues.

Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide.

Low-voltage-activated or T-type Ca(2+) channels (T-channels) are widely expressed, especially in the central nervous system where they contribute to pacemaker activities and are involved in the pathogenesis of epilepsy. Proper elucidation of their cellular functions has been hampered by the lack of selective pharmacology as well as the absence of generic endogenous regulations. We report here that both cloned (alpha(1G), alpha(1H) and alpha(1I) subunits) and native T-channels are blocked by the endogenous cannabinoid, anandamide. Anandamide, known to exert its physiological effects through cannabinoid receptors, inhibits T-currents independently from the activation of CB1/CB2 receptors, G-proteins, phospholipases and protein kinase pathways. Anandamide appears to be the first endogenous ligand acting directly on T-channels at submicromolar concentrations. Block of anandamide membrane transport by AM404 prevents T-current inhibition, suggesting that anandamide acts intracellularly. Anandamide preferentially binds and stabilizes T-channels in the inactivated state and is responsible for a significant decrease of T-currents associated with neuronal firing activities. Our data demonstrate that anandamide inhibition of T-channels can regulate neuronal excitability and account for CB receptor-independent effects of this signaling molecule.

The alpha1I T-type calcium channel exhibits faster gating properties when overexpressed in neuroblastoma/glioma NG 108-15 cells.

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion.

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.

Molecular and functional properties of the human alpha(1G) subunit that forms T-type calcium channels.

We describe here several novel properties of the human alpha(1G) subunit that forms T-type calcium channels. The partial intron/exon structure of the corresponding gene CACNA1G was defined and several alpha(1G) isoforms were identified, especially two isoforms that exhibit a distinct III-IV loop: alpha(1G-a) and alpha(1G-b). Northern blot and dot blot analyses indicated that alpha(1G) mRNA is predominantly expressed in the brain, especially in thalamus, cerebellum, and substantia nigra. Additional experiments have also provided evidence that alpha(1G) mRNA is expressed at a higher level during fetal life in nonneuronal tissues (i.e. kidney, heart, and lung). Functional expression in HEK 293 cells of a full-length cDNA encoding the shortest alpha(1G) isoform identified to date, alpha(1G-b), resulted in transient, low threshold activated Ca(2+) currents with the expected permeability ratio (I(Sr) > I(Ca) >/= I(Ba)) and channel conductance ( approximately 7 pS). These properties, together with slowly deactivating tail currents, are typical of those of native T-type Ca(2+) channels. This alpha(1G)-related current was inhibited by mibefradil (IC(50) = 2 microM) and weakly blocked by Ni(2+) ions (IC(50) = 148 microM) and amiloride (IC(50) > 1 mM). We showed that steady state activation and inactivation properties of this current can generate a "window current" in the range of -65 to -55 mV. Using neuronal action potential waveforms, we show that alpha(1G) channels produce a massive and sustained Ca(2+) influx due to their slow deactivation properties. These latter properties would account for the specificity of Ca(2+) influx via T-type channels that occurs in the range of physiological resting membrane potentials, differing considerably from the behavior of other Ca(2+) channels.

In vitro and in vivo characterization of a calcium modulator of the diphenylalkylamine type with selective coronary dilatory properties.

UNLABELLED: VUF 8929 (N-¿2-[bis(p-fluorophenyl)methoxy]ethyl¿-(2-phenyl)ethylamine maleate, CAS 140890-71-7) is a diphenylalkylamine derivative structurally related to prenylamine. The calcium antagonistic properties of this compound have been studied in in vitro and in vivo systems. VUF 8929 has affinity for the voltage-operated calcium channel. Its pKD for the displacement of [3H]nitrendipine bound to cerebral rat cortex is 6.27 (+/- 0.17). The compound influences the [3H]nitrendipine binding through an allosteric interaction with a site adjacent to the dihydropyridine binding site. Competitive experiments with the additional presence of the phenylalkylamine gallopamil showed that this allosteric site is a property common to diphenyl- and phenylalkylamines. It was further observed that VUF 8929 has a high affinity for calmodulin as it shows high potency in inhibiting the calmodulin mediated activation of PDE. The inhibition of K+ (IC50 0.5 mumol/l)- and noradrenaline (IC50 1.3 mumol/l)-induced contractions of rabbit aorta rings was in the same concentration range as found for the calmodulin inhibitory activity. In vitro platelet aggregation was also inhibited in the same concentration range when washed platelets were used. Thus calmodulin antagonism may contribute to the observed effects on aorta ring contractions and platelets aggregation. Platelet aggregation, however, in media in which albumin was added or in platelet rich plasma was not affected. It is assumed that due to the high lipophilicity, common to many diphenylalkylamines, VUF 8929 has a strong binding to plasma proteins. This may also explain why orally administered VUF 8929 did not affect the alpha 2-induced pressor response in pithed rats and the ex vivo collagen induced aggregation response. The haemodynamic profile in anaesthetized dogs showed that intravenous injected VUF 8929 reduced the workload of the heart while coronary blood flow increases at a dose of 0.3 mg/kg. Reversible occlusion of the coronary artery, which leads to S-T segment elevation and local venous acidosis, were reduced by VUF 8929 indicating that the compound has anti-ischaemic properties. IN CONCLUSION: VUF 8929 is a calcium antagonist which has anti-ischaemic properties, reduces the workload of the heart and increases coronary flow. Due to these properties VUF 8929 is a potential cardioprotective agent.

[In tolerance reactions to Rheomacrodes. Apropos of 3 cases]. / Réactions d'intolérances au Rhéomacrodex. A propos de 3 observations

Three cases of reactions of intolerance to Rheomacrodex were observed during these last four years in our Department, and this led us to attempt to sum up their exact nature and in particular the possibility of an immuno-allergic orgin. From the clinical standpoint, the symptomatology is dominated by the combination of abrupt collapse associated with Quincke's edema in two cases, and with bronchospasm in the third. The course was rapidly favourable under respiratory and circulatory emergency resuscitation. The immuno-allergic enquiry carried out in one case, did not enable one to demonstrate the responsibility of an antigen-antibody reaction. Analysis of the different works appeared on this subject, in particular those of the Swedish school, of W. RICHTER et col., did not enable one to pick out with any certainty the responsibility of an antigen-antibody conflict in the genesis of these reactions.