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.

Relativistic electron generation in interactions of a 30 TW laser pulse with a thin foil target.

Energy and angular distributions of the fast outgoing electron beam induced by the interaction of a 1 J, 30 fs, 2 x 10(19) W/cm(2), 10 Hz laser with a thin foil target are characterized by electron energy spectroscopy and photonuclear reactions. We have investigated the effect of the target thickness and the intensity contrast ratio level on the electron production. Using a 6-microm polyethylene target, up to 4 x 10(8) electrons with energies between 5 and 60 MeV were produced per laser pulse and converted to gamma rays by bremsstrahlung in a Ta secondary target. The rates of photofission of U as well as photonuclear reactions in Cu, Au, and C samples have been measured. In optimal focusing conditions, about 0.06% of the laser energy has been converted to outgoing electrons with energies above 5 MeV. Such electrons leave the target in the laser direction with an opening angle of 2.5 degrees.

[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.

A portable transfer chamber for electrochemical measurements on electrodes prepared in ultra-high vacuum.

This paper describes a versatile, light weight, and portable chamber dedicated to the transfer of electrodes from ultra-high vacuum (UHV) to atmospheric pressure and the liquid phase. This chamber can be connected to a liquid-phase reaction cell to perform electrochemical measurements and transfer back the electrode to the UHV environment. The experimental set-up can also be turned in order to make the electrode the bottom of the electrochemical cell. The validity and the efficiency of the experimental set-up were tested with a Pt(111) surface that provides unique electrochemical features in acidic sulphate-containing solution. This transfer chamber concept provides the surface science community with a new and versatile tool, complementary to existing systems, which allows fast electrolyte purging or electrochemical measurements under well-controlled mass transport conditions.

Improvements and new capabilities for the multiple Knudsen cell device used in high-temperature mass spectrometry.

The thermodynamic properties of condensed phases, i.e. the activities of components, can be determined from partial pressures measured by the Knudsen cell mass spectrometric method. Improvements in accuracy and yield of this method are obtained with the use of twin cells, an idea proposed in the 1960s. The multiple cell method was perfected in 1977 in our laboratory. Changes to molecular beam sampling and furnace assemblies were required to make the multiple Knudsen cell technique work properly. This paper summarizes these prerequisites, and presents a new device and the associated method of measurements, as well as the necessary tests performed with a silver sample in each cell.

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.

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.

Specific properties of T-type calcium channels generated by the human alpha 1I subunit.

We have cloned and expressed a human alpha(1I) subunit that encodes a subtype of T-type calcium channels. The predicted protein is 95% homologous to its rat counterpart but has a distinct COOH-terminal region. Its mRNA is detected almost exclusively in the human brain, as well as in adrenal and thyroid glands. Calcium currents generated by the functional expression of human alpha(1I) and alpha(1G) subunits in HEK-293 cells were compared. The alpha(1I) current activated and inactivated approximately 10 mV more positively. Activation and inactivation kinetics were up to six times slower, while deactivation kinetics was faster and showed little voltage dependence. A slower recovery from inactivation, a lower sensitivity to Ni(2+) ions (IC(50) approximately 180 micrometer), and a larger channel conductance (approximately 11 picosiemens) were the other discriminative features of the alpha(1I) current. These data demonstrate that the alpha(1I) subunit encodes T-type Ca(2+) channels functionally distinct from those generated by the human alpha(1G) or alpha(1H) subunits and point out that human and rat alpha(1I) subunits have species-specific properties not only in their primary sequence, but also in their expression profile and electrophysiological behavior.

Low-energy measurement of the 7Be(p,gamma)8B cross section.

We have measured the cross section of the 7Be(p,gamma)8B reaction for E(c.m.) = 185.8, 134.7, and 111.7 keV using a radioactive 7Be target (132 mCi). Single and coincidence spectra of beta+ and alpha particles from 8B and 8Be* decay, respectively, were measured using a large acceptance spectrometer. The zero energy S factor inferred from these data is 18.5+/-2.4 eV b and a weighted mean value of 18.8+/-1.7 eV b (theoretical uncertainty included) is deduced when combining this value with our previous results at higher energies.

Electron acceleration by a wake field forced by an intense ultrashort laser pulse.

Plasmas are an attractive medium for the next generation of particle accelerators because they can support electric fields greater than several hundred gigavolts per meter. These accelerating fields are generated by relativistic plasma waves-space-charge oscillations-that can be excited when a high-intensity laser propagates through a plasma. Large currents of background electrons can then be trapped and subsequently accelerated by these relativistic waves. In the forced laser wake field regime, where the laser pulse length is of the order of the plasma wavelength, we show that a gain in maximum electron energy of up to 200 megaelectronvolts can be achieved, along with an improvement in the quality of the ultrashort electron beam.