Ivan Kadurin

The Increased Trafficking of the Calcium Channel Subunit α2δ-1 to Presynaptic Terminals in Neuropathic Pain Is Inhibited by the α2δ Ligand Pregabalin

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 an in vivo rat 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 PGB in vivo is 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 α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins, a posttranslational modification essential for function

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 α2δ subunit; both of these subunits influence the trafficking and properties of CaV1 and CaV2 channels. The α2δ 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 α2δ 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 α2δ subunits studied, α2δ-1, α2δ-2, and α2δ-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 α2δ 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 α2δ subunits. We also show that GPI anchoring of α2δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α2δ subunits are coexpressed. In conclusion, this study redefines our understanding of α2δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.

Presynaptic HCN1 channels regulate CaV3.2 activity and neurotransmission at select cortical synapses

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 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 are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (CaV3.2) Ca2+ channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and CaV3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

A new look at calcium channel α2δ subunits

The classical roles of α(2)δ proteins are as accessory calcium channel subunits, enhancing channel trafficking. They were thought to have type-I transmembrane topology, but we find that they can form GPI-anchored proteins. Moreover α(2)δ-1 and α(2)δ-3 have been shown to have novel functions in synaptogenesis, independent of their effect on calcium channels. In neurons, the α(2)δ-1 subunits are present mainly in presynaptic terminals. Peripheral sensory nerve injury results in the up-regulation of α(2)δ-1 in dorsal root ganglion (DRG) neurons, and there is a consequent increase in trafficking of α(2)δ-1 to their terminals. Furthermore, gabapentinoid drugs, which bind to α(2)δ-1 and α(2)δ-2, not only impair their trafficking, but also affect α(2)δ-1-dependent synaptogenesis. These drugs may interfere with α(2)δ function at several different levels.

Functional exofacially tagged N-type calcium channels elucidate the interaction with auxiliary α2δ-1 subunits

Significance The auxiliary α2δ-1 subunits of voltage-gated calcium (CaV) channels are important therapeutic targets, representing the receptor for gabapentinoid drugs in neuropathic pain therapy. It is therefore important to understand their function. Because α2δ subunits augment calcium currents, it is believed that they increase cell-surface expression of these channels. Here, using exofacially tagged CaV2.2 constructs, we now show this to be the case. However, recent proteomic analysis found that α2δ subunits are associated only loosely and nonquantitatively with CaV2 channels, challenging their role as calcium channel subunits. In contrast, we find that CaV2.2 and α2δ-1 are intimately and completely associated at the plasma membrane and that this is not disrupted by the α2δ-1 ligand gabapentin, which reduces cell-surface expression of both CaV2.2 and α2δ-1. CaV1 and CaV2 voltage-gated calcium channels are associated with β and α2δ accessory subunits. However, examination of cell surface-associated CaV2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged CaV2 channels. Here we report the development of fully functional CaV2.2 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 α2δ subunits copurify with CaV2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α2δ subunits are associated with these channels at the cell surface. We found that, whereas α2δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with CaV2.2/β1b, despite our finding that α2δ-1 increases plasma-membrane CaV2.2 expression. However, this was due to occlusion of the antigenic epitope by association with CaV2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α2δ-1 and the α1 subunit at the plasma membrane. We further show that, although CaV2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α2δ-1 (but not a gabapentin-insensitive α2δ-1 mutant), the interaction between CaV2.2 and α2δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that CaV2.2 and α2δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction.

Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo- and Heteromeric Channels, and Identification of Regions Important for Activation by H+

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H+-gated Na+ channels, ASIC4 is not gated by H+, and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H+, zASIC4.2 is not. This differential response to H+ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4.1. Surface expression of zASIC4.1 was much increased by formation of heteromeric channels, suggesting that zASIC4.1 contributes to heteromeric ASICs in zebrafish neurons. Robust surface expression of H+-insensitive zASIC4.2 suggests that zASIC4.2 functions as a homomer and is gated by an as yet unknown stimulus, different from H+. Moreover, we identified a small region just distal to the first transmembrane domain that is crucial for the differential H+ response of the two paralogs. This post-TM1 domain may have a general role in gating of members of this gene family.

CALCIUM CURRENTS ARE ENHANCED BY α2δ-1 LACKING ITS MEMBRANE ANCHOR

Background: We examined the role of membrane anchoring of voltage-gated calcium channel α2δ subunits. Results: We used a truncated α2δ-1 construct (α2δ-1ΔC-term), which still increases CaV2.1/β1b currents, despite being mainly secreted. Conclusion: The effect of α2δ-1ΔC-term on calcium currents does not involve secretion and subsequent re-binding to the plasma membrane. Significance: C-terminal membrane anchoring of α2δ is not essential for calcium current enhancement. The accessory α2δ subunits of voltage-gated calcium channels are membrane-anchored proteins, which are highly glycosylated, possess multiple disulfide bonds, and are post-translationally cleaved into α2 and δ. All α2δ subunits have a C-terminal hydrophobic, potentially trans-membrane domain and were described as type I transmembrane proteins, but we found evidence that they can be glycosylphosphatidylinositol-anchored. To probe further the function of membrane anchoring in α2δ subunits, we have now examined the properties of α2δ-1 constructs truncated at their putative glycosylphosphatidylinositol anchor site, located before the C-terminal hydrophobic domain (α2δ-1ΔC-term). We find that the majority of α2δ-1ΔC-term is soluble and secreted into the medium, but unexpectedly, some of the protein remains associated with detergent-resistant membranes, also termed lipid rafts, and is extrinsically bound to the plasma membrane. Furthermore, heterologous co-expression of α2δ-1ΔC-term with CaV2.1/β1b results in a substantial enhancement of the calcium channel currents, albeit less than that produced by wild-type α2δ-1. These results call into question the role of membrane anchoring of α2δ subunits for calcium current enhancement.

Thrombospondin-4 reduces binding affinity of [(3)H]-gabapentin to calcium-channel α2δ-1-subunit but does not interact with α2δ-1 on the cell-surface when co-expressed.

The α2δ proteins are auxiliary subunits of voltage-gated calcium channels, and influence their trafficking and biophysical properties. The α2δ ligand gabapentin interacts with α2δ-1, and inhibits calcium channel trafficking. However, α2-1 has also been proposed to play a synaptogenic role, independent of calcium channel function. In this regard, α2δ-1 was identified as a ligand of thrombospondins, with the interaction involving the thrombospondin synaptogenic domain and the α2δ-1 von-Willebrand-factor domain. Co-immunoprecipitation between α2δ-1 and the synaptogenic domain of thrombospondin-2 was prevented by gabapentin. We therefore examined whether interaction of thrombospondin with α2δ-1 might reciprocally influence 3H-gabapentin binding. We concentrated on thrombospondin-4, because, like α2δ-1, it is upregulated in neuropathic pain models. We found that in membranes from cells co-transfected with α2δ-1 and thrombospondin-4, there was a Mg2+ -dependent reduction in affinity of 3H-gabapentin binding to α2δ-1. This effect was lost for α2δ-1 with mutations in the von-Willebrand-factor-A domain. However, the effect on 3H-gabapentin binding was not reproduced by the synaptogenic EGF-domain of thrombospondin-4. Partial co-immunoprecipitation could be demonstrated between thrombospondin-4 and α2δ-1 when co-transfected, but there was no co-immunoprecipitation with thrombospondin-4-EGF domain. Furthermore, we could not detect any association between these two proteins on the cell-surface, indicating the demonstrated interaction occurs intracellularly.

Proteolytic maturation of α2δ represents a checkpoint for activation and neuronal trafficking of latent calcium channels

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

LRP1 influences trafficking of N-type calcium channels via interaction with the auxiliary α2δ-1 subunit.

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.