7 3 a r t I C l e SThe level of expression of voltage-gated calcium channels at the plasma membrane is a key regulator of calcium homeostasis in excitable cells, and of downstream effects such as calcium-dependent transcription 1,2 . Members of the high voltage-activated (HVA) calcium channel family are heteromultimeric protein complexes that contain a pore-forming α 1 subunit that defines the channel subtype, plus ancillary α 2 -δ and β subunits that not only alter the function of the α 1 subunit but also regulate the trafficking of the channel complex to the plasma membrane 3-8 . The mammalian genome encodes four different types of Cavβ subunit that have distinct spatial and temporal expression patterns [4][5][6] . With the exception of Cavβ 2a , these subunits are cytoplasmic proteins that physically bind to a region in the α 1 subunit domain I-II linker that is highly conserved among all HVA calcium channels and is termed the alpha interaction domain (AID) 7 . Crystal structure data show that the Cavβ subunit contains interacting SH3 and guanylate kinase domains, with the latter participating in high-affinity binding to the AID region [8][9][10] . The physiological consequences of this interaction are underscored by gene knockout studies showing that deletion of the Cavβ 1a or Cavβ 2a subunits causes embryonic lethality 11,12 and by findings that a premature stop mutation in Cavβ 4 causes an epileptic phenotype in mice 13 .It has been suggested that the Cavβ subunit masks an endoplasmic reticulum retention signal on the Cav2.1 α 1 subunit 14 , thereby leading to increased cell surface expression of P/Q-type channels. However, no specific endoplasmic reticulum retention motif in the AID and surrounding regions of the α 1 subunit has been identified, and removing the AID motif in the I-II linker of Cav2.1 does not increase current amplitude in the absence of Cavβ (ref. 15). Moreover, it is not clear whether different HVA calcium channel isoforms share common retention motifs. Here we show that Cav1.2 (L-type) calcium channels contain an endoplasmic reticulum retention motif in the proximal C-terminal region, and we provide evidence that the Cavβ subunit has a role in regulating proteasomal degradation of these channels. Our data show that the Cavβ subunit acts as a molecular switch that prevents the ubiquitination of the channels and their targeting to the ERAD complex and thereby regulates their expression at the plasma membrane. RESULTS Cavb regulates endoplasmic reticulum retention of Cav1.2We first performed an ELISA assay involving a Cav1.2 channel construct tagged with an extracellular hemagglutinin (HA) epitope (Fig. 1a). We compared immunoluminescence between permeabilized and nonpermeabilized cells, which allowed us to quantify the relative proportion of Cav1.2 channels that were inserted into the plasma membrane. Coexpression with the Cavβ 1b or Cavβ 2a subunit mediated a significant increase in the fraction of Cav1.2 channels at the cell surface (Fig. 1a and data not shown). This was confirmed by HA...

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

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Necrosis, as opposed to apoptosis, is recognized as a nonspecific cell death that induces tissue inflammation and is preceded by cell edema. In non-neuronal cells, the latter has been explained by defective outward pumping of Na ؉ caused by metabolic depletion or by increased Na ؉ influx via membrane transporters. Here we describe a novel mechanism of swelling and necrosis; namely the influx of Na

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