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...

N -methyl- d -aspartate receptors (NMDARs) mediate critical CNS functions, whereas excessive activity contributes to neuronal damage. At physiological glycine concentrations, NMDAR currents recorded from cultured rodent hippocampal neurons exhibited strong desensitization in the continued presence of NMDA, thus protecting neurons from calcium overload. Reducing copper availability by specific chelators (bathocuproine disulfonate, cuprizone) induced nondesensitizing NMDAR currents even at physiologically low glycine concentrations. This effect was mimicked by, and was not additive with, genetic ablation of cellular prion protein (PrP C ), a key copper-binding protein in the CNS. Acute ablation of PrP C by enzymatically cleaving its cell-surface GPI anchor yielded similar effects. Biochemical studies and electrophysiological measurements revealed that PrP C interacts with the NMDAR complex in a copper-dependent manner to allosterically reduce glycine affinity for the receptor. Synthetic human Aβ 1–42 (10 nM–5 μM) produced an identical effect that could be mitigated by addition of excess copper ions or NMDAR blockers. Taken together, Aβ 1–42 , copper chelators, or PrP C inactivation all enhance the activity of glycine at the NMDAR, giving rise to pathologically large nondesensitizing steady-state NMDAR currents and neurotoxicity. We propose a physiological role for PrP C , one that limits excessive NMDAR activity that might otherwise promote neuronal damage. In addition, we provide a unifying molecular mechanism whereby toxic species of Aβ 1–42 might mediate neuronal and synaptic injury, at least in part, by disrupting the normal copper-mediated, PrP C -dependent inhibition of excessive activity of this highly calcium-permeable glutamate receptor.

Background: CRMP2 is an axonal guidance protein that has been linked to NMDA receptor-mediated excitotoxicity. Results: A CRMP2 peptide protects against NMDA receptor-mediated excitotoxicity in vitro and in vivo following a traumatic brain injury. Conclusion: CRMP2 is a novel target for neuroprotection. Significance: Targeting CRMP2 could lead to development of neurotherapeutics against traumatic brain injury as well as other neuronal insults.

We have investigated the heterodimerization of ORL1 receptors and classical members of the opioid receptor family. All three classes of opioid receptors could be co-immunoprecipitated with ORL1 receptors from both transfected tsA-201 cell lysate and rat dorsal root ganglia lysate, suggesting that these receptors can form heterodimers. Consistent with this hypothesis, in cells expressing either one of the opioid receptors together with ORL1, prolonged ORL1 receptor activation via nociceptin application resulted in internalization of the opioid receptors. Conversely, -, ␦-, and -opioid receptor activation with the appropriate ligands triggered the internalization of ORL1. The -opioid receptor/ORL1 receptor heterodimers were shown to associate with N-type calcium channels, with activation of -opioid receptors triggering N-type channel internalization, but only in the presence of ORL1. Furthermore, the formation of opioid receptor/ORL1 receptor heterodimers attenuated the ORL1 receptor-mediated inhibition of N-type channels, in part because of constitutive opioid receptor activity. Collectively, our data support the existence of heterodimers between ORL1 and classical opioid receptors, with profound implications for effectors such as N-type calcium channels.ORL1 (opioid receptor-like 1) receptors (also known as NOP or nociceptin receptors) belong to the class of G␣ i/o -linked seven-helix transmembrane receptors (1, 2). They are structurally related to members of the classical opioid receptor family (i.e. -, ␦-, and -opioid receptors) but do not interact with known opioid receptor agonists or antagonists. Instead, they are activated by the endogenous ligand orphanin FQ (also known as nociceptin), a 17-amino acid polypeptide (3). ORL1 receptors are expressed in both the central and peripheral nervous systems; the physiological effects of receptor activation include stimulation of food intake, reduced anxiety, reduced withdrawal symptoms, and when activated peripherally, analgesia (4 -6). There is a functional cross-talk between ORL1 receptors and -opioid receptors such that chronic administration of the -receptor agonist morphine results in increased ORL1 receptor expression levels (7), whereas knock-out of the ORL1 receptor gene results in decreased morphine tolerance (8) without compensatory changes in opioid receptor expression (9). This may hint at the possibility of overlapping mechanisms controlling cellular expression levels of these two receptor subtypes.We have recently shown that ORL1 receptors physically interact with N-type calcium channels and that this interaction results in two distinct consequences: first, an agonist-independent inhibition of N-type channels due to constitutive receptor activity (10), and second, receptor-mediated trafficking of N-type channels to and from the plasma membrane (11). Neither of these phenomena appeared to occur with -opioid receptors (11). ORL1 receptors have also been shown to heterodimerize with -opioid receptors (12), with dimerized receptors showing altered sensitiv...

N -Methyl-D-aspartate (NMDA) receptors mediate a wide range of important nervous system functions. Conversely, excessive NMDA receptor activity leads to cytotoxic calcium overload and neuronal damage in a wide variety of CNS disorders. It is well established that NMDA receptors are tightly regulated by a number of cell signalling pathways. Recently, it has been shown that NMDA receptor activity is modulated by cellular prion protein (PrP C ) in a copper-dependent manner. Here we give an overview of the current state of knowledge concerning the novel concept of potent modulation of this receptor's kinetics by copper ions, and the interplay between NMDA receptors and PrP C in the context of neurological diseases such as Alzheimer's disease, epilepsy, pain and depression.