The α2δ-like Protein Cachd1 Increases N-type Calcium Currents and Cell Surface Expression and Competes with α2δ-1

摘要

class="head no_bottom_margin" id="sec1title">IntroductionVoltage-gated calcium (CaV) channels are key constituents of excitable cells, including muscles, neurons, and secretory cells, and are essential for their function (for a review, see ). The neuronal N-type (CaV2.2) and P/Q-type (CaV2.1) channels are critical for presynaptic release of neurotransmitters (for a review, see ), with N-type calcium channels playing a particularly important role in primary afferent neurotransmission involving pain pathways (for a review, see ). CaV α1 subunits form the pore of the channels, determining their main biophysical and pharmacological properties (), but the associated β and α2δ proteins represent auxiliary subunits that are important contributors to the trafficking and biophysical properties of the channel complexes (, , , ). The β subunits increase CaV currents by binding to the intracellular I-II linker (), promoting folding (), hyperpolarizing current activation (), preventing polyubiquitination (), and inhibiting proteasomal degradation (, ).By contrast, the mechanism by which the α2δ subunits increase trafficking and function of channel complexes is less well understood (, href="#bib6" rid="bib6" class=" bibr popnode">Cassidy et al., 2014, href="#bib12" rid="bib12" class=" bibr popnode">Ferron et al., 2018, href="#bib20" rid="bib20" class=" bibr popnode">Kadurin et al., 2016, href="#bib36" rid="bib36" class=" bibr popnode">Savalli et al., 2016). The α2δ-1 subunit, in combination with neuronal calcium channels, is the therapeutic target for gabapentinoid drugs, used for the alleviation of neuropathic pain conditions and as an add-on therapy in certain epilepsies (href="#bib13" rid="bib13" class=" bibr popnode">Field et al., 2006), and it is therefore important to understand its mechanism of action. The α2δ proteins undergo several post-translational processing steps, including N-glycosylation, proteolytic cleavage into α2 and δ (href="#bib8" rid="bib8" class=" bibr popnode">De Jongh et al., 1990, href="#bib10" rid="bib10" class=" bibr popnode">Ellis et al., 1988, href="#bib18" rid="bib18" class=" bibr popnode">Jay et al., 1991), and glycosyl-phosphatidylinositol (GPI) anchoring (href="#bib7" rid="bib7" class=" bibr popnode">Davies et al., 2010).The recent structure of the skeletal muscle CaV1.1 complex (href="#bib48" rid="bib48" class=" bibr popnode">Wu et al., 2016) has revealed a complex interaction of α2δ-1 with several extracellular loops in domains I-III of CaV1.1. In the present study, we have taken advantage of the insights provided by this structure to probe the role of the von Willebrand factor A (VWA) domain and investigate whether there is a role for other α2δ domains in CaV channel function. In previous studies, by mutating the metal ion-dependent adhesion site (MIDAS) motif in the VWA domain of α2δ subunits, we have shown that the VWA domains of both α₂δ-1 and α₂δ-2 are key to promoting calcium channel trafficking and function (href="#bib5" rid="bib5" class=" bibr popnode">Cantí et al., 2005, href="#bib6" rid="bib6" class=" bibr popnode">Cassidy et al., 2014, href="#bib16" rid="bib16" class=" bibr popnode">Hoppa et al., 2012). The structure confirms the interaction of the MIDAS motif with the Ca_V1.1 α1 subunit (href="#bib48" rid="bib48" class=" bibr popnode">Wu et al., 2016). However, we also found that mutating the MIDAS motif reduced the trafficking of α₂δ-1 itself when it was expressed alone (href="#bib6" rid="bib6" class=" bibr popnode">Cassidy et al., 2014). In the present study, we have therefore taken the reciprocal step of mutating the residue in Ca_V2.2 with which α₂δ-1 is predicted to bind to examine whether other regions, such as their Cache domains, play a role in promoting Ca_V2.2 trafficking and function. The Cache domains in α₂δ-1, which have homology to domains in bacterial chemotaxis receptors (href="#bib2" rid="bib2" class=" bibr popnode">Anantharaman and Aravind, 2000), have also been shown to interact with the Ca_V1.1 α1 subunit (href="#bib48" rid="bib48" class=" bibr popnode">Wu et al., 2016). We have compared the effect of α₂δ-1 with that of Cachd1, identified bioinformatically to be related to α₂δ proteins (href="#bib47" rid="bib47" class=" bibr popnode">Whittaker and Hynes, 2002). Cachd1 has a VWA domain with a disrupted MIDAS motif but retains multiple predicted Cache domains. Surprisingly, we found that expression of Cachd1 increased both Ca_V2.2 currents and cell surface trafficking in both cell lines and neurons. By contrast, expression of Cachd1 did not increase the closely related Ca_V2.1 currents, indicating that this effect shows specificity for certain calcium channels. Furthermore, Cachd1 competed with α₂δ-1 for binding to Ca_V2.2 and for its functional effects and can therefore inhibit responses to α₂δ-1.