A new look at calcium channel α2δ subunits

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The classical roles of α2δ 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 α2δ-1 and α2δ-3 have been shown to have novel functions in synaptogenesis, independent of their effect on calcium channels. In neurons, the α2δ-1 subunits are present mainly in presynaptic terminals. Peripheral sensory nerve injury results in the up-regulation of α2δ-1 in dorsal root ganglion (DRG) neurons, and there is a consequent increase in trafficking of α2δ-1 to their terminals. Furthermore, gabapentinoid drugs, which bind to α2δ-1 and α2δ-2, not only impair their trafficking, but also affect α2δ-1-dependent synaptogenesis. These drugs may interfere with α2δ function at several different levels.

Introduction

Voltage-gated calcium (CaV) channels are heteromeric proteins consisting of three or four subunits. The pore-forming subunit of CaV channels is the α1 subunit, and this determines the main biophysical and pharmacological properties of the channels (Figure 1). For the low-voltage-activated calcium channels (CaV3 or T-type channels), they appear to be able to function well as monomers. However, for the high-voltage activated (HVA) CaV1 and CaV2 subfamilies, the α1 subunit is generally thought to be associated with a membrane-anchored, predominantly extracellular, α2δ subunit (for reviews see [1, 2]), and a cytoplasmic CaVβ subunit (Figure 1). Four α2δ subunits have been cloned: α2δ-1 [3], α2δ-2 [4] α2δ-3 [5] and α2δ-4 [6]. This review will cover several novel findings concerning these proteins, in particular with respect to their topology, and their roles in synaptogenesis as well as in calcium channel function.

Section snippets

Novel topology and post-translational processing of α2δ subunits

The topological organization of the α2δ protein was first determined for α2δ-1, and has been thought to be similar in all the four α2δ subunits that have been cloned (for reviews see [1, 2]). The α2 subunit is disulfide-bonded to a smaller δ subunit, as determined using biochemical studies on purified dihydropyridine receptors from skeletal muscle which were examined under reducing and non-reducing conditions [7, 8, 9]. However, once the cDNA sequence was obtained it became clear that both α2

Evidence that α2δ subunits are GPI-anchored, rather than transmembrane proteins

All α2δ subunits have an N-terminal signal or leader sequence that directs the protein into the lumen of the endoplasmic reticulum at the time of translation, where this signal sequence is cleaved (Figure 2a). The α2δ subunits also have a C-terminal hydrophobic domain that has led them to be considered as type I trans-membrane proteins. However, this hydrophobic region is rather short, predicted to be between 17 and 22 amino acids (Figure 2a), and is followed by either only one or two

Membrane localization, trafficking and endocytosis of α2δ subunits

GPI-anchoring has major implications for the structure and function of α2δ proteins, but it also affects their membrane localization, their intracellular trafficking and their endocytosis, since these proteins cannot signal across the lipid bilayer to interact directly with sorting proteins [21, 22]. Moreover it explains the marked localization of α2δ proteins in a detergent-resistant membrane (DRM) fraction, also termed ‘lipid rafts’, that we have noted previously [23]. Lipid raft localization

Function of α2δ subunits in calcium channel complexes

The α2δ subunits enhance the forward trafficking of the α1 calcium channel subunits and decrease their turnover at the plasma membrane. Moreover they also influence the biophysical properties of the channels (for reviews see [1, 2]). Their loss, as in the naturally occurring α2δ-2 knockout strains of mice, ducky and ducky2J [32], results in a reduction in calcium channel currents in Purkinje neurons, where α2δ-2 is normally strongly expressed, and has marked effects on Purkinje cell function [33

Presynaptic localization and trafficking of α2δ subunits

A recent immunohistochemical study has shown very clearly that in the central nervous system, α2δ-1 is mainly associated with terminal fields, rather than cell bodies [39]. With regard to α2δ-3, where similar immunohistochemical studies are lacking, mutations in the Drosophila α2δ-3 subunit have revealed it to be the likely binding partner for the cacophony calcium channel that is involved in presynaptic vesicle release and calcium channel expression at active zones [40]. This suggests that α2

Novel functions of α2δ subunits in muscle function and synaptogenesis

Although α2δ proteins have been clearly defined as subunits of voltage-gated calcium channels, other roles for these proteins unrelated to calcium channel function have also been proposed. One suggestion that α2δ subunits may have multiple roles comes from the finding that the genes for both α2δ-2 [4] and α2δ-3 [43] have been potentially implicated in tumour susceptibility and growth. It has also been found that in developing skeletal muscle the α2δ-1 subunits were segregated from CaV1.1,

Mechanism of action of the gabapentinoid drugs

A further key feature of the α2δ-1 and α2δ-2 subunits is that they bind the anti-epileptic and anti-allodynic drugs gabapentin and pregabalin. It has recently been shown that binding of these drugs to the α2δ-1 subunit is essential for their therapeutic effect in neuropathic pain [51], namely a reduction of excitatory transmission in the spinal cord. However, most reports indicate that these drugs produce very little acute inhibition of calcium currents [2, 29, 52]. In contrast, we showed that

Conclusions

The α2δ proteins are classically known as accessory calcium channel subunits. They have been described as type 1 trans-membrane proteins, but our recent research suggests that they are GPI-anchored, which has implications for their intracellular trafficking and endocytosis. The α2δ-1 subunits are strongly up-regulated in DRG neurons in experimental models of neuropathic pain, and this protein is trafficked from the cell bodies to the presynaptic terminals in the spinal cord. The localization of

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This study describes work to which many members of our group, past and present have contributed. We thank the Medical Research Council (UK) and the Biotechnology and Biological Sciences Research Council, UK (BBSRC) for support of the work described from the authors’ laboratory.

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