Synchrony of spontaneous calcium activity in mouse neocortex before synaptogenesis.

Spontaneous calcium activity can be detected in embryonic mouse cortical slices as fluorescence intensity variations, in the presence of a fluorescent calcium indicator. Current methods to detect and quantify these variations depend heavily on experimenters whose judgement may interfere with measurement. In the present work, we developed new software called CalSignal for automatic detection and tracking of cellular bodies and quantification of spontaneous calcium activity on time-series of confocal fluorescence images. Analysis of 28 neocortical slices revealed that 21.0% of detected cells displayed peaks of fluorescence corresponding to spontaneous activity, with a mean frequency of one peak per 4 min. This activity was blocked in the absence of extracellular calcium but was not modified after depletion of calcium stores with thapsigargin or blockade of voltage-gated calcium channels with Ni2+. Further, statistical analysis of calcium activity revealed concomitant activation of distant cells in 24 slices, and the existence of a significant network of synchrony based on such coactivations in 17 slices out of 28. These networks enclosed 84.3% of active cells, scattered throughout the neocortical wall (mean distance between cellular bodies, 111.7 microm). Finally, it was possible to identify specific cells which were synchronously active with more neighbouring cells than others. The identity of these nodal cells remains to be investigated to fully comprehend the role of spontaneous calcium activity, before synaptogenesis, in shaping cortical neurogenesis.

The CD38-independent ADP-ribosyl cyclase from mouse brain synaptosomes: a comparative study of neonate and adult brain.

cADPR (cADP-ribose), a metabolite of NAD+, is known to modulate intracellular calcium levels and to be involved in calcium-dependent processes, including synaptic transmission, plasticity and neuronal excitability. However, the enzyme that is responsible for producing cADPR in the cytoplasm of neural cells, and particularly at the synaptic terminals of neurons, remains unknown. In the present study, we show that endogenous concentrations of cADPR are much higher in embryonic and neonate mouse brain compared with the adult tissue. We also demonstrate, by comparing wild-type and Cd38-/- tissues, that brain cADPR content is independent of the presence of CD38 (the best characterized mammalian ADP-ribosyl cyclase) not only in adult but also in developing tissues. We show that Cd38-/- synaptosome preparations contain high ADP-ribosyl cyclase activities, which are more important in neonates than in adults, in line with the levels of endogenous cyclic nucleotide. By using an HPLC method and adapting the cycling assay developed initially to study endogenous cADPR, we accurately examined the properties of the synaptosomal ADP-ribosyl cyclase. This intracellular enzyme has an estimated K(m) for NAD+ of 21 microM, a broad optimal pH at 6.0-7.0, and the concentration of free calcium has no major effect on its cADPR production. It binds NGD+ (nicotinamide-guanine dinucleotide), which inhibits its NAD+-metabolizing activities (K(i)=24 microM), despite its incapacity to cyclize this analogue. Interestingly, it is fully inhibited by low (micromolar) concentrations of zinc. We propose that this novel mammalian ADP-ribosyl cyclase regulates the production of cADPR and therefore calcium levels within brain synaptic terminals. In addition, this enzyme might be a potential target of neurotoxic Zn2+.

Early expression of sodium channel transcripts and sodium current by cajal-retzius cells in the preplate of the embryonic mouse neocortex.

In mouse, the first neurons are generated at embryonic day (E) 12 and form the preplate (PP), which contains a mix of future marginal zone cells, including Cajal-Retzius cells, and subplate cells. To detect developmental changes in channel populations in these earliest-generated neurons of the cerebral cortex, we studied the electrophysiological properties of proliferative cells of the ventricular zone and postmitotic neurons of the PP at E12 and E13, using whole-cell patch-clamp recordings. We found an inward sodium current in 55% of PP cells. To determine whether sodium currents occur in a specific cell type, we stained recorded cells with an antibody for calretinin, a calcium-binding protein found specifically in Cajal-Retzius cells. All calretinin-positive cells had sodium currents, although so did some calretinin-negative cells. To correlate the Na current expression to Na channel gene expression with the Cajal-Retzius cell phenotype, we performed single-cell reverse transcription-PCR on patch-clamp recorded cells to detect expression of the Cajal-Retzius cell marker reelin and the Na channel isoforms SCN 1, 2, and 3. These results showed that virtually all Cajal-Retzius cells (97%), as judged by reelin expression, express the SCN transcript identified as the SCN3 isoform. Of these, 41% presented a functional Na current. There is, however, a substantial SCN-positive population in the PP (27% of SCN-positive cells) that does not express reelin. These results raise the possibility that populations of pioneer neurons of the PP, including Cajal-Retzius cells, gain neuronal physiological properties early in development via expression of the Na(v)1.3 (SCN3) Na channel isoform.

Evidence for an intracellular ADP-ribosyl cyclase/NAD+-glycohydrolase in brain from CD38-deficient mice.

Cyclic ADP-ribose, a metabolite of NAD+, is known to modulate intracellular calcium levels and signaling in various cell types, including neural cells. The enzymes responsible for producing cyclic ADP-ribose in the cytoplasm of mammalian cells remain unknown; however, two mammalian enzymes that are capable of producing cyclic ADP-ribose extracellularly have been identified, CD38 and CD157. The present study investigated whether an ADP-ribosyl cyclase/NAD+-glycohydrolase independent of CD38 is present in brain tissue. To address this question, NAD+ metabolizing activities were accurately examined in developing and adult Cd38-/- mouse brain protein extracts and cells. Low ADP-ribosyl cyclase and NAD+-glycohydrolase activities (in the range of pmol of product formed/mg of protein/min) were detected in Cd38-/- brain at all developmental stages studied. Both activities were found to be associated with cell membranes. The activities were significantly higher in Triton X-100-treated neural cells compared with intact cells, suggesting an intracellular location of the novel cyclase. The cyclase and glycohydrolase activities were optimal at pH 6.0 and were inhibited by zinc, properties which are distinct from those of CD157. Both activities were enhanced by guanosine 5'-O-(3-thiotriphosphate), a result suggesting that the novel enzyme may be regulated by a G protein-dependent mechanism. Altogether our results indicate the presence of an intracellular membrane-bound ADP-ribosyl cyclase/NAD+-glycohydrolase distinct from CD38 and from CD157 in mouse brain. This novel enzyme, which is more active in the developing brain than in the adult tissue, may play an important role in cyclic ADP-ribose-mediated calcium signaling during brain development as well as in adult tissue.

Human skeletal muscle triadin: gene organization and cloning of the major isoform, Trisk 51.

We obtained the gene organization of human triadin gene by aligning the DNA coding sequence of human 95-kDa triadin (Trisk 95) with human genomic database. We identified a novel human triadin isoform, a potential human homologue of rat Trisk 51. We show that both isoforms of triadin, Trisk 51 and Trisk 95, are alternative splice variants of the same gene. We demonstrated experimentally the existence of this Trisk 51 transcript in human skeletal muscle and cloned its full length cDNA. We further demonstrated that the protein encoded by this transcript is expressed in the human skeletal muscle. In addition, unlike other species, Trisk 51 is the major triadin isoform expressed in human skeletal muscle, whereas Trisk 95 is below the detection level in the two types of muscles tested.

CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain.

CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue.

Functional interaction between mouse spermatogenic LVA and thapsigargin-modulated calcium channels.

The acrosome reaction in mouse is triggered by a long-lasting calcium signaling produced by a chain of openings of several calcium channels, a low-voltage-activated (LVA) calcium channel, an inositol trisphosphate receptor (IP(3)R), and the store-operated calcium channel TRP2. Since mature sperm cells are refractory to patch clamp experiments, we study the functional interactions among those sperm calcium channels in spermatogenic cells. We have studied the role of cytosolic calcium in voltage-dependent facilitation of low voltage-activated calcium channels. Calcium concentration was modified through the inclusion of the calcium buffers, EGTA and BAPTA, in the recording pipette solution, and by addition of calcium modulators like thapsigargin and the calcium ionophore A23187. We demonstrate that lowering calcium concentration below resting level allows to evidence a voltage-dependent facilitation. We also show that LVA calcium channels present strong voltage-dependent inhibition by thapsigargin. This effect is independent of cytosolic calcium elevation secondary to calcium store depletion and to the activation of TRP channels. Our data evidence an interesting functional relationship, in this cell type, between LVA channels and proteins whose activity is related to calcium filling state of the endoplasmic reticulum (presumably TRP channels and inositol triphosphate receptor). These relationships may contribute to the regulation of calcium signaling during acrosome reaction of mature sperm cell.

Multiple determinants in voltage-dependent P/Q calcium channels control their retention in the endoplasmic reticulum.

Surface expression level of voltage-dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage-activated calcium channels requires the association of the pore-forming subunit, Cavalpha, with the auxiliary subunit, Cavbeta. In the absence of this auxiliary subunit, Cavalpha is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8 alpha chimeras, the molecular determinants of Cavalpha2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as 'ER retention'). This study demonstrates that the I-II loop of Cavalpha2.1 contains multiple ER-retention determinants beside the beta subunit association domain. In addition, the I-II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I-II loop, the N- and C-termini of Cavalpha2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low-threshold calcium channels, the I-II loop of Cavalpha1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i). a correct folding of Cavalpha in order to mask several internal ER-retention determinants and (ii). the association of other proteins, including the Cavbeta subunit, to suppress the remaining ER-retention determinants.

Modelling of the III-IV loop, a domain involved in calcium channel Ca(v)2.1 inactivation, highlights a structural homology with the gamma subunit of G proteins.

We have modelled the conformation of the III-IV loop of the Ca(v)2.1 subunit of P/Q calcium channels, a loop that is implicated in fast voltage-dependent inactivation. Change in channel inactivation requires its direct interaction with the I-II loop. This interaction occurs with an affinity in the order of 70 nm. Intracellular injection of a 40-mer III-IV loop-derived peptide produces an increase in the rate of fast inactivation. This alteration in channel kinetic is also accompanied by a hyperpolarizing shift in the steady-state voltage-dependence of inactivation. None of these effects are observed in the presence of a beta subunit, suggesting the existence of a competitive mechanism of action between the beta subunit and the III-IV loop. Amino acid sequence comparison using BLAST reveals that the III-IV loop shares 53% identity with the gamma subunit of G proteins. Because of the pivotal contribution of the III-IV loop to inactivation, an atomic model of the III-IV loop was generated by both homology modelling and molecular mechanics calculations. Using the X-ray structures of the betagamma dimer of the heterotrimeric G-proteins as templates, the III-IV loop is predicted to contain a well-structured alpha-helix at the amino-terminus with both the N- and C-termini having the same orientation in the plane of the inner lipid bilayer. We provide a hypothetical working model in which we propose that the III-IV loop interacts with the I-II loop via its Gbetagamma binding domain.

Use of a purified and functional recombinant calcium-channel beta4 subunit in surface-plasmon resonance studies.

Native high-voltage-gated calcium channels are multi-subunit complexes comprising a pore-forming subunit Ca(v) and at least two auxiliary subunits alpha(2)delta and beta. The beta subunit facilitates cell-surface expression of the channel and contributes significantly to its biophysical properties. In spite of its importance, detailed structural and functional studies are hampered by the limited availability of native beta subunit. Here, we report the purification of a recombinant calcium-channel beta(4) subunit from bacterial extracts by using a polyhistidine tag. The purified protein is fully functional since it binds on the alpha1 interaction domain, its main Ca(v)-binding site, and regulates the activity of P/Q calcium channel expressed in Xenopus oocytes in a similar way to the beta(4) subunit produced by cRNA injection. We took advantage of the functionality of the purified material to (i) develop an efficient surface-plasmon resonance assay of the interaction between two calcium channel subunits and (ii) measure, for the first time, the affinity of the recombinant His-beta(4) subunit for the full-length Ca(v)2.1 channel. The availability of this purified material and the development of a surface-plasmon resonance assay opens two immediate research perspectives: (i) drug screening programmes applied to the Ca(v)/beta interaction and (ii) crystallographic studies of the calcium-channel beta(4) subunit.

The interaction between the I-II loop and the III-IV loop of Cav2.1 contributes to voltage-dependent inactivation in a beta -dependent manner.

We have investigated the molecular mechanisms whereby the I-II loop controls voltage-dependent inactivation in P/Q calcium channels. We demonstrate that the I-II loop is localized in a central position to control calcium channel activity through the interaction with several cytoplasmic sequences; including the III-IV loop. Several experiments reveal the crucial role of the interaction between the I-II loop and the III-IV loop in channel inactivation. First, point mutations of two amino acid residues of the I-II loop of Ca(v)2.1 (Arg-387 or Glu-388) facilitate voltage-dependent inactivation. Second, overexpression of the III-IV loop, or injection of a peptide derived from this loop, produces a similar inactivation behavior than the mutated channels. Third, the III-IV peptide has no effect on channels mutated in the I-II loop. Thus, both point mutations and overexpression of the III-IV loop appear to act similarly on inactivation, by competing off the native interaction between the I-II and the III-IV loops of Ca(v)2.1. As they are known to affect inactivation, we also analyzed the effects of beta subunits on these interactions. In experiments in which the beta(4) subunit is co-expressed, the III-IV peptide is no longer able to regulate channel inactivation. We conclude that (i) the contribution of the I-II loop to inactivation is partly mediated by an interaction with the III-IV loop and (ii) the beta subunits partially control inactivation by modifying this interaction. These data provide novel insights into the mechanisms whereby the beta subunit, the I-II loop, and the III-IV loop altogether can contribute to regulate inactivation in high voltage-activated calcium channels.

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) antagonizes this effect. Two peptides, covering these sequences (peptide A(Sk) and C(Sk), respectively) were immobilized on polystyrene beads. We demonstrate that peptide A(Sk) binds to the skeletal isoform of RyR (RyR1) whereas peptide C(Sk) does not. Using surface plasmon resonance detection, we show that 1) domain Thr(671)-Leu(690) is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A(Sk) with RyR1 is not modulated by Ca(2+) (pCa 9-2) nor by Mg(2+) (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC(50) = 10 nM) and inhibited by both rapamycin (IC(50) = 5 nM) and FK506. Peptide A(Sk) induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A(Sk) on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR alpha(1)-subunit corresponding to Thr(671)-Leu(690) and show that the association of FKBP12 with RyR1 specifically modulates this interaction.