Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit ␣ 2 ␦-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both ␣ 2 ␦ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of ␣ 2 ␦-1 in DRG neurons, both at the light-and electron-microscopic level. We found that, on the side of the ligation, ␣ 2 ␦-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of ␣ 2 ␦-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of ␣ 2 ␦-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of ␣ 2 ␦-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of ␣ 2 ␦-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

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All auxiliary ␣2␦ subunits of voltage-gated Ca 2؉ (CaV) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in ␣2␦-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the ␣2␦-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant ␣2␦-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT ␣2␦-2, the MIDAS mutant ␣2␦-2 subunit did not enhance and, in some cases, further diminished Ca V1.2, -2.1, and -2.2 currents coexpressed with ␤1b by using either Ba 2؉ or Na ؉ as a permeant ion. Furthermore, expression of the MIDAS mutant ␣2␦-2 reduced surface expression and strongly increased the perinuclear retention of Ca V␣1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108 -15 cells. Despite the presence of endogenous ␣2␦ subunits, heterologous expression of ␣2␦-2 in differentiated NG108 -15 cells further enhanced the endogenous high-threshold Ca 2؉ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that ␣2␦ subunits normally interact with the CaV␣1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the ␣2␦ subunit is required to promote trafficking of the ␣1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.integrin ͉ neuron ͉ motif ͉ expression V oltage-gated Ca 2ϩ (Ca V ) channels are composed of a poreforming ␣1 subunit that determines the main biophysical properties of the channel. For the Ca V 1 and -2 subfamilies, this subunit is associated with an intracellular ␤ subunit (for review, see refs. 1 and 2) and a membrane-anchored, predominantly extracellular ␣ 2 ␦ subunit (for review, see ref.3). Mammalian genes encoding 10 ␣1, 4 ␤, and 4 ␣ 2 ␦ subunits have been identified (for reviews, see refs. 2 and 4). The topology of the ␣ 2 ␦ protein has been determined in detail only for ␣ 2 ␦-1 but is thought to generalize to all 4 ␣ 2 ␦ subunits (for review, see ref.3). All ␣ 2 ␦ subunits have predicted N-terminal signal sequences, indicating that the N terminus is extracellular. In early studies of ␣ 2 ␦-1 purified from skeletal and cardiac muscle, it was determined that the ␣ 2 subunit is disulfide-bonded to a transmembrane ␦ subunit, and both subunits are the products of a single gene, encoding the ␣ 2 ␦ protein, that is posttranslationally cleaved into ␣ 2 and ␦ (5).Subsequent to the identification of ␣ 2 ␦ subunits as stoichiometric components of skeletal muscle Ca 2ϩ channels, ␣ 2 ␦ subunits have also been shown to be associated with native cardiac (L-type) (6) and neuronal N-and P͞Q-type channels (7,8). In coexpression stud...

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α 2 δ subunit; both of these subunits influence the trafficking and properties of Ca V 1 and Ca V 2 channels. The α 2 δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α 2 δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α 2 δ subunits studied, α 2 δ-1, α 2 δ-2, and α 2 δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositolphospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α 2 δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α 2 δ subunits. We also show that GPI anchoring of α 2 δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α 2 δ subunits are coexpressed. In conclusion, this study redefines our understanding of α 2 δ subunits, both in terms of their role in calciumchannel function and other roles in synaptogenesis.lipid raft | posttranslational | electrophysiology | immunocytochemistry

SummaryAnti-CD40 monoclonal antibodies (mAbs) that promote or inhibit receptor function hold promise as therapeutics for cancer and autoimmunity. Rules governing their diverse range of functions, however, are lacking. Here we determined characteristics of nine hCD40 mAbs engaging epitopes throughout the CD40 extracellular region expressed as varying isotypes. All mAb formats were strong agonists when hyper-crosslinked; however, only those binding the membrane-distal cysteine-rich domain 1 (CRD1) retained agonistic activity with physiological Fc gamma receptor crosslinking or as human immunoglobulin G2 isotype; agonistic activity decreased as epitopes drew closer to the membrane. In addition, all CRD2-4 binding mAbs blocked CD40 ligand interaction and were potent antagonists. Thus, the membrane distal CRD1 provides a region of choice for selecting CD40 agonists while CRD2-4 provides antagonistic epitopes.