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.

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

Ca V 1 and Ca V 2 voltage-gated calcium channels are associated with β and α 2 δ accessory subunits. However, examination of cell surface-associated Ca V 2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α 2 δ subunits copurify with Ca V 2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α 2 δ subunits are associated with these channels at the cell surface. We found that, whereas α 2 δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with Ca V 2.2/β1b, despite our finding that α 2 δ-1 increases plasmamembrane Ca V 2.2 expression. However, this was due to occlusion of the antigenic epitope by association with Ca V 2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α 2 δ-1 and the α 1 subunit at the plasma membrane. We further show that, although Ca V 2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α 2 δ-1 (but not a gabapentin-insensitive α 2 δ-1 mutant), the interaction between Ca V 2.2 and α 2 δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that Ca V 2.2 and α 2 δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction. P urification of L-type voltage-gated calcium (Ca V ) channels from skeletal muscle shows that they consist of a pore-forming α 1 subunit, Ca V 1.1, associated with three accessory subunits, β1, α 2 δ-1, and γ 1 (1, 2). Cardiac L-type channels have a similar subunit composition, although the α 1 subunit is α1C and the γ subunit is not present (3). However, the study of cell surface-associated N-type (Ca V 2.2) and P/Q-type (Ca V 2.1) calcium channels has been hampered by the lack both of antibodies to cell-surface epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the cell surface, in both cell lines and neurons. Using this methodological advance, we can now examine directly the effect of the auxiliary subunits on cellsurface expression of Ca V 2 channels.Although α 2 δ subunits have been shown to be associated with Ca V 2.1 and Ca V 2.2 following purification (4, 5), it has recently been suggested that the α 2 δ subunits are associated only very loosely and nonquantitatively with Ca V 2 channels (6), calling into question their role as calcium channel subunits. This study found that the α 2 δ proteins α 2 δ-1, α 2 δ-2, and α 2 δ-3 c...

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they play an important role in regulating synaptic potential integration and plasticity. Here, we demonstrate that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. We found that HCN channels inhibit glutamate synaptic release by suppressing the activity of low threshold voltage-gated T- (CaV3.2) type Ca2+ channels. In agreement, electron microscopy showed the co-localisation of pre-synaptic HCN1 and CaV3.2 subunit. This represents a novel mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes.DOI: http://dx.doi.org/10.7554/eLife.21143.001