Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Cav1.2 channels.

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits ß2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor ß2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)ß and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)ß subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)ß at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.

PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane.

Phosphatidylinositol 3-kinase (PI3K) has been shown to enhance native voltage-dependent calcium channel (Ca(v)) currents both in myocytes and in neurons; however, the mechanism(s) responsible for this regulation were not known. Here we show that PI3K promotes the translocation of GFP-tagged Ca(v) channels to the plasma membrane in both COS-7 cells and neurons. We show that the effect of PI3K is mediated by Akt/PKB and specifically requires Ca(v)beta(2) subunits. The mutations S574A and S574E in Ca(v)beta(2a) prevented and mimicked, respectively, the effect of PI3K/Akt-PKB, indicating that phosphorylation of Ser574 on Ca(v)beta(2a) is necessary and sufficient to promote Ca(v) channel trafficking.

Comparison of human embryomorphokinetic parameters in sequential or global culture media.

A prospective study on randomized patients was conducted to determine how morphokinetic parameters are altered in embryos grown in sequential versus global culture media. Eleven morphokinetic parameters of 160 single embryos transferred were analyzed by time lapse imaging involving two University-affiliated in vitro fertilization (IVF) centers. We found that the fading of the two pronuclei occurred earlier in global (22.56±2.15 hpi) versus sequential media (23.63±2.71 hpi; p=0.0297). Likewise, the first cleavage started earlier at 24.52±2.33 hpi vs 25.76±2.95 hpi (p=0.0158). Also, the first cytokinesis was shorter in global medium, lasting 18±10.2 minutes in global versus 36±37.8 minutes in sequential culture medium (p <0.0001). We also observed a significant shortening in the duration of the 2-cell stage in sequential medium: 10.64 h±2.75 versus 11.66 h±1.11 in global medium (p=0.0225) which suggested a faster progression of the embryos through their first mitotic cell cycle. In conclusion, morphokinetic analysis of human embryos by Time lapse imaging reveals significant differences in five kinetic variables according to culture medium. Our study highlights the need to adapt morphokinetic analysis accordingly to the type of media used to best support human early embryo development.

[Assisted oocyte activation: a new tool for severe male factor infertility treatment]. / Aide à l'activation ovocytaire - Un nouvel atout dans le traitement des infertilités masculines sévères.

In severe male infertility, in vitro fertilization (IVF) with intra-cytoplasmic sperm injection (ICSI) represents the sole available therapeutic option. However this technique is not always successful in promoting fertilization, as some couples completely and repeatedly fail to obtain any embryo. In many cases, this failure can be attributed to a defective rise in intracellular calcium, which is required to achieve oocyte activation. Over the last twenty years, several laboratories dedicated to assisted reproduction technologies have been using a calcium ionophore to assist oocyte activation. The aim of this review is to give an overview of the advances and consequences associated with this new technique referred to as assisted oocyte activation.

Constitutive PtdIns(3,4,5)P3 synthesis promotes the development and survival of early mammalian embryos.

Mammalian preimplantation embryos develop in the oviduct as individual entities, and can develop and survive in vitro, in defined culture media lacking exogenous growth factors or serum. Therefore, early embryos must generate intrinsic signals that promote their development and survival. In other cells, activation of class I phosphoinositide 3-kinase (PI3K) is a universal mechanism to promote cell proliferation and survival. Here, we examined whether PI3K is intrinsically activated during preimplantation development. Using GFP-tagged pleckstrin homology domains to monitor PtdIns(3,4,5)P(3) synthesis, we show that PI3K is constitutively activated in mouse preimplantation embryos. E-cadherin ligation promotes PtdIns(3,4,5)P(3) synthesis at sites of blastomere adhesion at all cleavage stages. In addition, in culture conditions that promote autocrine signalling, a second pool of PtdIns(3,4,5)P(3) is generated in the apical membrane of early stage blastomeres. We show that constitutive PtdIns(3,4,5)P(3) synthesis is necessary for optimal development to blastocyst and to prevent large-scale apoptosis at the time of cavitation.

Mechanism of action of Gq to inhibit G beta gamma modulation of CaV2.2 calcium channels: probed by the use of receptor-G alpha tandems.

The stable interaction of a G-protein coupled receptor and a particular partner G-protein was made possible by creating tandems between the alpha(2A) adrenergic receptor (alpha(2A)-R) and pertussis toxin-resistant mutants of different G alpha subunits of heterotrimeric G-proteins. Both alpha(2A)-R-G alpha(o) and alpha(2A)-R-G alpha(i) proved able to reconstitute agonist-induced voltage-dependent inhibition of N-type calcium channels (Ca(V)2.2) similar to the wild-type alpha(2A)-R when expressed in COS-7 cells. The interaction of G(q) with the G(i/o) signaling pathways was studied by expressing either G alpha(q) or a chimeric construct based on G alpha(q) containing the last five amino acids of G alpha(z), which is activated by alpha(2A)-R. It was found that G alpha(qz5) activated by the wild-type alpha(2A)-R inhibited Ca(V)2.2 currents in a voltage-independent fashion. Furthermore, G alpha(qz5) counteracted the voltage-dependent inhibition resulting from alpha(2A)-R-G alpha(o) activation. We subsequently investigated the basis for the behavior of G alpha(qz5). Our evidence suggests that this occurs as a result of a downstream effect of activation of G alpha(qz5) because it was blocked by C-terminal construct of phospholipase C beta 1. Furthermore it is likely to occur in part via protein kinase C (PKC) activation, because the PKC activator phorbol dibutyrate mimicked the effects of G alpha(qz5) in alpha(2A)-R-G alpha(o)-transfected cells. Conversely, cells expressing both alpha(2A)-R-G alpha(o) and G alpha(qz5) exhibited a partial restoration of voltage-dependent inhibition in the presence of the PKC inhibitor bisindolylmaleimide I (GF 109203X). The potential sites of phosphorylation are discussed.

The novel product of a five-exon stargazin-related gene abolishes Ca(V)2.2 calcium channel expression.

We have cloned and characterized a new member of the voltage-dependent Ca(2+) channel gamma subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential gamma subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated gamma(7). Expression of human gamma(7) has been localized specifically to brain. N-type current through Ca(V)2.2 channels was almost abolished when co-expressed transiently with gamma(7) in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that gamma(7) has this effect by causing a large reduction in expression of Ca(V)2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed gamma(7) was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like gamma subunits, different gene structure and the unique functional properties of gamma(7) imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.