Abstract
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.
Highlights
The a2d subunits of voltage-gated calcium channels (CaV) have been identified to be key proteins in synaptic function and synaptogenesis (Dickman et al, 2008; Kurshan et al, 2009; Hoppa et al, 2012; Eroglu et al, 2009; Saheki and Bargmann, 2009)
Transient expression in cell lines of wild type (WT) a2d-1 resulted in only partial cleavage into a2 and d subunits, clearly observed only following deglycosylation (Figure 1c, lanes 1 and 3), compared to the complete proteolytic cleavage observed in brain (Figure 1c, lanes 2 and 4)
Using extracellularly-tagged CaV2.2 to quantify the channels inserted into the plasma membrane (Cassidy et al, 2014), we found that in undifferentiated N2A cells, the uncleaved a2(3C)d-1 remains capable of increasing the cell surface density of CaV2.2 channels (123% increase compared to CaV2.2/b1b alone), by a similar extent to WT a2d-1 (140% increase; Figure 2d,e). a2(3C)d-1 increased cell surface expression of CaV2.2 in the tsA-201 cells used for electrophysiology (Figure 2—figure supplement 3)
Summary
The a2d subunits of voltage-gated calcium channels (CaV) have been identified to be key proteins in synaptic function and synaptogenesis (Dickman et al, 2008; Kurshan et al, 2009; Hoppa et al, 2012; Eroglu et al, 2009; Saheki and Bargmann, 2009). A single gene encodes each a2d protein, which undergoes several post-translational processing steps, including proteolytic cleavage into disulfide-linked a2 and d (Davies et al, 2010; Jay et al, 1991; Ellis et al, 1988; De Jongh et al, 1990). Both a2 and d have been shown previously to be important for the function of a2d-1 to increase CaV currents and influence the biophysical properties of the currents (Gurnett et al, 1996, 1997).
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