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Neuronal N-type (CaV2.2) voltage-gated calcium channels are essential for neurotransmission from primary afferent terminals in the dorsal horn. In this study we have utilized a knock-in mouse expressing CaV2.2 with an inserted extracellular hemagglutinin-tag (CaV2.2_HA), to visualise the distribution of endogenous CaV2.2 in dorsal root ganglion (DRG) neurons and their primary afferents in the dorsal horn. We examined the effect of partial sciatic nerve ligation (PSNL) and found an increase in CaV2.2_HA only in large and medium dorsal root ganglion neurons, and also in deep dorsal-horn synaptic terminals. Furthermore, there is a parallel increase in co-expression with GFRα1, present in a population of low threshold mechanoreceptors, both in large DRG neurons and in their terminals. The increased expression of CaV2.2_HA in these DRG neurons and their terminals is dependent on the presence of the auxiliary subunit α2δ-1, which is required for channel trafficking to the cell surface and to synaptic terminals, and likely contributes to enhanced synaptic transmission at these synapses following PSNL. In contrast the increase of GFRα1 is not altered in α2δ-1 knockout mice. We also found following PSNL there is patchy loss of glomerular synapses immunoreactive for CaV2.2_HA and CGRP or IB4, restricted to the superficial layers of the dorsal horn. This reduction is not dependent on α2δ-1, and likely reflects partial deafferentation of C-nociceptor presynaptic terminals. Therefore, we can distinguish in this pain model two different events affecting specific DRG terminals, with opposite consequences for CaV2.2_HA expression and function in the dorsal horn.

The auxiliary α

Voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The CaV1 and CaV2 channels are associated with auxiliary β- and α2δ-subunits. The molecular mechanisms involved in α2δ subunit trafficking, and the effect of α2δ subunits on trafficking calcium channel complexes remain poorly understood. Here we show that α2δ-1 is a ligand for the Low Density Lipoprotein (LDL) Receptor-related Protein-1 (LRP1), a multifunctional receptor which mediates trafficking of cargoes. This interaction with LRP1 is direct, and is modulated by the LRP chaperone, Receptor-Associated Protein (RAP). LRP1 regulates α2δ binding to gabapentin, and influences calcium channel trafficking and function. Whereas LRP1 alone reduces α2δ-1 trafficking to the cell-surface, the LRP1/RAP combination enhances mature glycosylation, proteolytic processing and cell-surface expression of α2δ-1, and also increase plasma-membrane expression and function of CaV2.2 when co-expressed with α2δ-1. Furthermore RAP alone produced a small increase in cell-surface expression of CaV2.2, α2δ-1 and the associated calcium currents. It is likely to be interacting with an endogenous member of the LDL receptor family to have these effects. Our findings now provide a key insight and new tools to investigate the trafficking of calcium channel α2δ subunits.

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

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 phosphatidylinositol-phospholipase 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 calcium-channel function and other roles in synaptogenesis.

Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit α2δ-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 α2δ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used anin vivorat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of α2δ-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, α2δ-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 α2δ-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of α2δ-1 mRNA and protein in DRGs.In vitro, PGB reduced plasma membrane expression of α2δ-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGBin vivois associated with impaired anterograde trafficking of α2δ-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.

The mechanism of action of the antiepileptic and antinociceptive drugs of the gabapentinoid family has remained poorly understood. Gabapentin (GBP) binds to an exofacial epitope of the α 2 δ-1 and α 2 δ-2 auxiliary subunits of voltage-gated calcium channels, but acute inhibition of calcium currents by GBP is either very minor or absent. We formulated the hypothesis that GBP impairs the ability of α 2 δ subunits to enhance voltage-gated Ca 2+ channel plasma membrane density by means of an effect on trafficking. Our results conclusively demonstrate that GBP inhibits calcium currents, mimicking a lack of α 2 δ only when applied chronically, but not acutely, both in heterologous expression systems and in dorsal root-ganglion neurons. GBP acts primarily at an intracellular location, requiring uptake, because the effect of chronically applied GBP is blocked by an inhibitor of the system-L neutral amino acid transporters and enhanced by coexpression of a transporter. However, it is mediated by α 2 δ subunits, being prevented by mutations in either α 2 δ-1 or α 2 δ-2 that abolish GBP binding, and is not observed for α 2 δ-3, which does not bind GBP. Furthermore, the trafficking of α 2 δ-2 and Ca V 2 channels is disrupted both by GBP and by the mutation in α 2 δ-2, which prevents GBP binding, and we find that GBP reduces cell-surface expression of α 2 δ-2 and Ca V 2.1 subunits. Our evidence indicates that GBP may act chronically by displacing an endogenous ligand that is normally a positive modulator of α 2 δ subunit function, thereby impairing the trafficking function of the α 2 δ subunits to which it binds.

The mouse mutant ducky and its allele ducky2Jrepresent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations inCacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). TheCacna2d2du2Jmutation results in a 2 bp deletion in the coding region and a complete loss of α2δ-2 protein. Here we show thatdu2J/du2Jmice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C,du2J/du2JPCs show no spontaneous intrinsic activity.Du2J/du2Jmice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Althoughdu2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However,du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

All auxiliary α 2 δ subunits of voltage-gated Ca 2+ (Ca V ) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α 2 δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α 2 δ-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 α 2 δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α 2 δ-2, the MIDAS mutant α 2 δ-2 subunit did not enhance and, in some cases, further diminished Ca V 1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba 2+ or Na + as a permeant ion. Furthermore, expression of the MIDAS mutant α 2 δ-2 reduced surface expression and strongly increased the perinuclear retention of Ca V α1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α 2 δ subunits, heterologous expression of α 2 δ-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 α 2 δ subunits normally interact with the Ca V α1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α 2 δ subunit is required to promote trafficking of the α1 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.

The CaVβ subunits of voltage-gated calcium channels regulate these channels in several ways. Here we investigate the role of these auxiliary subunits in the expression of functional N-type channels at the plasma membrane and in the modulation by G-protein-coupled receptors of this neuronal channel. To do so, we mutated tryptophan 391 to an alanine within the α-interacting domain (AID) in the I-II linker of CaV2.2. We showed that the mutation W391 virtually abolishes the binding of CaVβ1b and CaVβ2a to the CaV2.2 I-II linker and strongly reduced current density and cell surface expression of both CaV2.2/α2δ-2/β1b and/β2a channels. When associated with CaVβ1b, the W391A mutation also prevented the CaVβ1b-mediated hyperpolarization of CaV2.2 channel activation and steady-state inactivation. However, the mutated CaV2.2W391A/β1b channels were still inhibited to a similar extent by activation of the D2dopamine receptor with the agonist quinpirole. Nevertheless, key hallmarks of G-protein modulation of N-type currents, such as slowed activation kinetics and prepulse facilitation, were not observed for the mutated channel. In contrast, CaVβ2a was still able to completely modulate the biophysical properties of CaV2.2W391A channel and allow voltage-dependent G-protein modulation of CaV2.2W391A. Additional data suggest that the concentration of CaVβ2a in the proximity of the channel is enhanced independently of its binding to the AID by its palmitoylation. This is essentially sufficient for all of the functional effects of CaVβ2a, which may occur via a second lower-affinity binding site, except trafficking the channel to the plasma membrane, which requires interaction with the AID region.

Jasmonates (JAs) regulate Arabidopsis thaliana wound and defence responses, pollen development, and stress-related growth inhibition. Significantly, each of these responses requires COI1, an F-box protein. Other F-box proteins interact with SKP1 and cullin proteins to form SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination. To determine whether COI1 also functions in an SCF complex, we have characterized Arabidopsis proteins that bind to COI1. An Arabidopsis cDNA expression library was screened in yeast for clones that produce proteins which can bind to COI1. We recovered two SKP1 homologues and a histone deacetylase. The Arabidopsis F-box protein TIR1 interacted with SKP1 proteins, but not with the histone deacetylase. Mutant COI1 proteins revealed that the F-box is required for interaction with SKP1s, but that sequences in leucine-rich repeat domains are required for interaction with the histone deacetylase. Epitope-tagged COI1 was introduced into Arabidopsis plants and cell cultures. Co-immunoprecipitation experiments confirmed the interaction in planta of COI1 with SKP1-like proteins and histone deacetylase, and also indicated that COI1 interacted with cullin. These results suggest that COI1 forms an SCFCOI1 complex in vivo. COI1 is therefore expected to form a functional E3-type ubiquitin ligase in plants and to regulate expression of jasmonate responsive genes, possibly by targeted ubiquitination of a histone deacetylase.

The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1 , was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1−/−mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reducedin vivoelectrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1−/−mice, and α2δ-1−/−DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1−/−mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1−/−mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1−/−mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including β and α2δ, which modulate trafficking and functional properties of the channels.There are four known genes encoding α2δ subunits, which are believed to have similar structure. They consist of two peptides: the highly glycosylated α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. The α2 and δ peptides are encoded by a single gene as an uninterrupted α2δ pre-protein, which is further processed post-translationally. We have recently shown that α2δ subunits are glycosyl phophatidyl inositol (GPI)-achored proteins (Davies et al., 2010, PNAS 107:1654-1659), and this is essential for their function, and explains their localization in lipid raft fractions (Davies et al, 2006, J. Neurosci. 26: 8748-8757).We further studied the mechnism and consequences of the proteolytic cleavage of the α2δ subunits (α2δ-1, -2 and -3)and identified the cleavage sites in α2δ-2 and α2δ-3. Western blots from brain tissue revealed only the mature form of the protein strongly associated with lipid rafts. However, transfecting mammalian cells with cDNA for α2δ-1,-2,and -3 resulted in incomplete cleavage (∼ 40-60% processing) most probably due to limitations of the cleavage-mediating protease(s) in heterologous expression systems. The mature form is localized in lipid rafts, suggesting that maturation of the protein might occur in localized membrane domains. Moreover, electrophysiological recordings demonstrated that proteolytic cleavage is key to the function of these subunits.We are currently examining the nature of the protease(s) involved in this proteolytic processing.

Voltage-gated calcium channels (VGCCs) regulate calcium influx into all excitable cells. In the heart, the main calcium channels are the L-type VGCCs (LTCCs). These are localised to the sarcolemmal membrane, and are hetero-oligomeric complexes comprised of three non-covalently associated polypeptides; alpha1 (CaV1.2), alpha2delta and beta. We recently reported the 3D structure for a monomeric form of the cardiac LTCC1 using electron microscopy and single particle analysis. We also determined the first medium/low resolution structure of a T-type voltage gated calcium channel (CaV3.1) polypeptide. We identified the transmembrane and cytoplasmic domains of the T-type channel using labelling studies to determine the position of the C-terminus. By modelling of the CaV3.1 structure (comparable at these resolutions to CaV1.2) into the cardiac LTCC volume, we were able to delineate the subunit boundaries of the cardiac LTCC, leading to a proposal for a putative orientation of the LTCC with respect to the membrane bilayer. We have now extended these studies to include labelling of the extracellular alpha2 polypeptide using affinity purified antibodies raised against the Von Willebrand Factor A (VWA) domain and calmodulin-gold labelling of the C-terminus of CaV1.2. These data provide further support for the proposed orientation of the 3D structure of the cardiac LTCC.

Episodic ataxia 2 (EA2) is an autosomal dominant disorder caused by mutations in the gene CACNA1A that encodes the pore-forming CaV2.1 calcium channel subunit. The majority of EA2 mutations reported so far are nonsense or deletion/insertion mutations predicted to form truncated proteins. Heterologous expression of wild-type CaV2.1, together with truncated constructs that mimic EA2 mutants, significantly suppressed wild-type calcium channel function, indicating that the truncated protein produces a dominant-negative effect (Jouvenceau et al., 2001; Page et al., 2004). A similar finding has been shown for CaV2.2 (Raghib et al., 2001). We show here that a highly conserved sequence in the cytoplasmic N-terminus is involved in this process, for both CaV2.1 and CaV2.2 channels. Additionally, we were able to interfere with the suppressive effect of an EA2 construct by mutating key N-terminal residues within it. We postulate that the N-terminus of the truncated channel plays an essential part in its interaction with the full-length CaV2.1, which prevents the correct folding of the wild-type channel. In agreement with this, we were able to disrupt the interaction between EA2 and the full length channel by co-expressing a free N-terminal peptide.