Calcium channel diversity: multiple roles of calcium channel subunits

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Until recently we held the simple view that voltage-gated calcium channels consisted of an α1 subunit, usually associated with auxiliary β subunits and α2δ subunits and that skeletal muscle calcium channels were also associated with a γ subunit. However, as discussed here, there is now evidence that the auxiliary subunits may also perform other roles unrelated to voltage-gated calcium entry. In the past students were taught the simplistic view that second messenger signaling to voltage-gated calcium channels involved mainly phosphorylation of L-type calcium channels, Ca2+-dependent inactivation via calmodulin, and direct G-protein-mediated inhibition of the neuronal N and P/Q channels. However, it is now clear that there are many other means of modulating calcium channel activity, including receptor-mediated internalization, proteolytic cleavage, phosphorylation of β subunits, and interaction of calcium channels with other proteins, including enzymes masquerading as scaffold proteins.

Introduction

The pore-forming subunit of voltage-gated Ca2+ (Cav) channels is the α1 subunit, which determines the main biophysical and pharmacological properties of the channels (Figure 1). 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 review see [1]), and a cytoplasmic Cavβ subunit (for review see [2]) (Figure 1). Mammalian genes encoding 10 α1, four β and four α2δ subunits have been identified (Table 1), but the amount of complexity extends far beyond gene expression, through differential roles of various splice variants (for review see [3]), to the potential function of the Ca2+ channel accessory subunits in other capacities, and the postulated role of calmodulin (CaM) as an honorary calcium channel subunit.

The Cavβ and α2δ subunits are thought to play synergistic roles in trafficking Cav1 and Cav2 channels. The Cavβ subunits are believed to enhance the trafficking of the channels to the plasma membrane by binding via their guanylate kinase (GK)-like domain to the α interaction domain (AID) on the I–II linker of all the HVA α1 subunits (for review see [4]). It has been suggested that they act by facilitating the correct folding of the channels and promoting exit from the endoplasmic reticulum (ER) [5]. However, no specific ER retention signals have been identified on the I–II linker, and while the AID–GK domain interaction is intimately involved in trafficking and modulation of the channel complex, it is probably not the only domain of the β subunits to be implicated in this process [6, 7]. By contrast, the α2δ subunits promote trafficking of the calcium channel complex by a mechanism that involves their Von Willebrand factor-A (VWA) domain [8]. Although the exact site at which the α2δ subunits intervene in the trafficking process remains to be established, it is assumed that they interact with one or more exofacial domains of the channel.

Although the skeletal muscle calcium channel complex was identified, from the initial purification studies, to contain a γ subunit, the exact role of this protein (γ1) still remains unclear [9], and the function of the more recently cloned homologs, γ2–8, specifically as calcium channel subunits now seems unlikely. Indeed, γ2–8 have been identified to have a major role in AMPA glutamate receptor trafficking [10], and we have shown that although γ7 does affect calcium channel expression [11], it does so, not as a subunit, but via an effect on mRNA stability [12].

Section snippets

Novel forms of regulation of Cav channel surface expression and activity

There is an emerging theme throughout neurobiology that ion channels may be rapidly inserted into, and retrieved from the plasma membrane into endosomes, and that such trafficking processes may represent a rapid and reversible form of modulation. In now seems clear that the insertion and endocytosis of voltage-gated calcium channels may also occur with a rapid time course and in a regulated fashion. Furthermore, there may also be rapid control of calcium channel expression in pathological

Interaction of calcium channels directly with receptors to regulate cell surface expression

The existence of large macromolecular complexes, for example between calcium channels and receptors, enzymes or other channels, is an emerging theme. A classical route of inhibition of certain calcium channel subtypes (notably N and P/Q types) involves direct modulation by activation of G-protein-coupled receptors (GPCRs) via the βγ subunits, usually derived from Gi/o proteins (for review see [15]). However, it has recently been found that certain GPCRs, notably the dopamine D1 receptor,

Interaction of calcium channels with enzyme complexes to regulate their expression and properties

In cultured peptidergic bag cell neurons from Aplysia, it has been found that activation of protein kinase C (PKC) rapidly (within 10 minutes) recruits new Cav2 subunits to the plasma membrane of growth cones, via a process that involves actin [18]. However, the site of PKC phosphorylation remains unclear, and may not be the channel itself, but rather the process whereby Cav2-containing intracellular organelles are transported from the central core region within growth cones to the lamellipodia.

Modulation and processing in the C-terminus of Ca2+ channels

All HVA calcium channels have the ability to interact with CaM on their C-terminus, inducing both Ca2+-dependent inactivation (CDI) and facilitation, and the binding site involves variations on an isoleucine–glutamine-containing (IQ) domain (for review see [21]). However, Cav1.3 and Cav1.4 channels, despite having a great deal of sequence homology to Cav1.2 in the domains involved in CaM binding and CDI, are nevertheless involved in sustained transmitter release from cochlear hair cells and

Consequences of proteolytic processing of the C-terminus of Cav1.2

Native Cav1.2 has been shown to undergo proteolytic processing within the C-terminus, and the cleaved C-terminus, representing a 37–50 kDa polypeptide, contains the binding site for cyclic AMP-dependent protein kinase anchoring proteins (AKAPs), which tether protein kinase A (PKA) in the vicinity of the channel. The cleaved channel C-terminus and associated proteins are thought to remain attached to the rest of the channel, forming an autoinhibitory complex [25]. It is suggested that this may

Further roles of Cavβ subunits

The accessory Cavβ subunits, as well as playing key roles in trafficking and influencing the biophysical properties of calcium channels [2], have emerged as an important target for modulation, both via phosphorylation by PKA [31], CaMKII [32] and the PI3K/Akt pathway [33], and also by binding to a number of modulatory proteins. An example of the ability of second messenger pathways to influence channel trafficking processes was observed in by Viard et al. [33], showing that phosphorylation of

The role(s) of accessory α2δ subunits

The topology of the α2δ protein was first determined for α2δ-1, and is thought to generalize to all four α2δ subunits (for review see [1]). The exofacial α2 subunit was originally described as being disulfide-bonded to a trans-membrane δ subunit, although both subunits are actually the product of a single gene, encoding the α2δ pre-protein, which is post-translationally cleaved into α2 and δ. The α2δ subunits both enhance calcium channel expression and also influence the properties of the

Conclusion

I have outlined here some recent studies on the proteins that make up voltage-gated calcium channel heteromeric complexes, illustrating the intriguing complexity whereby they function. Both the pore-forming α1 subunits and the auxiliary β and α2δ subunits show degrees of sophistication in their roles, not envisaged when these channel complexes were first described. The β and α2δ subunits perform roles in calcium channel trafficking and also influence the biophysical properties of the channels.

References and recommended reading

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

  • • of special interest

  • •• of outstanding interest

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