Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

Abstract

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels.

Keywords: calcium channels; ion channels; protein complexes; subunit; voltage-dependent channels.

Figure 1

Structure and diversity of the α₁ pore-forming subunit of voltage-activated calcium channels. (A) Membrane topology of the α₁-subunit. Homologous to voltage-activated sodium channel, Ca_Vα₁ consists of four repeats (I–IV) joined by three cytoplasmic loops namely loop I–II, II–III, and III–IV. Loop I–II contains the highly conserved binding site for the β-subunit, referred to as α₁ interaction domain or AID. The C-terminus is relatively large and accommodates the binding site for Ca²⁺/calmodulin (IQ). Each repeat contains six transmembrane helices that include two separate structural domains: the voltage-sensing domain (VSD) formed by the first four helices and the pore domain (PD). (B) Three-dimensional structure of a tetrameric prokaryotic sodium channel (Na_VAB, PDB 3VRY) showing four peripheral VDS domains flanking a tetramer of PDs. Note that each VSD flanks the PD domain of a neighboring repeat. The central arrow points to the permeation pathway. (C) Ca_Vα₁ subunits and the corresponding type of Ca²⁺ current that they yield. Ca_V1.x and Ca_V2.x correspond to the channel subunit activated by large depolarization (high-voltage-activated, HVA). Ca_V3.x corresponds to channels activated by low voltages (LVA).

Figure 2

Structural organization and diversity of calcium channel α₂δ subunits. (A) Schematic representation of the domain arrangement of the four α₂δ isoforms identified in humans scaled according to the number of amino acids (α₂δ-1 acc. ·P54289; α₂δ-2 acc. Q9NY47; α₂δ-3 acc. Q8IZS8; α₂δ-4 acc. Q7Z3S7). The N-terminal signal peptide (SP) ranging from 19 amino acids for (α₂δ-2) to 29 residues for α₂δ-3 shown in red is not present in the mature protein. The blue segment depicts the WFA domain and the orange one the CACHE domain. (B) Ribbon diagram of the model proposed by Calderon-Rivera et al. (2012) showing the WFA domain in blue and CACHE in orange while the rest of the α₂ peptide is depicted in green. Within the δ peptide (cyan) an alpha helical segment (shown in red) is likely to represent a membrane-anchoring domain. The cysteine residues required for the α₂δ assembly are represented as van der Waals surfaces in yellow.

Figure 3

Gene family and structure of the β-subunit. (A) List of β-subunit encoding genes with splice variants (reviewed by Buraei and Yang, 2010). (B) Scheme of the α₁- and β-subunits showing the consensus amino acid sequence α₁-interaction domain or AID located within the intracellular I–II loop that binds to the β-subunit guanylate kinase domain (GK). (C) Domain organization and three-dimensional structure (PDB 1T3L) of the β-subunit. The SH3 domain is shown in orange and the GK domain in green. The AID peptide forms an alpha helix (shown in red) upon binding to the GK domain. The GK domain is responsible for modulation of function of HVA channels while SH3 appears to couple the channel to other cellular components.

Figure 4

Dendrogram and transmembrane topology of the γ subunit. (A) Phylogenetic dendrogram constructed using CLUSTAL to align CACNG sequences from vertebrates (zebra fish, human and Xenopus) and invertebrates. Proteins labeled stg nem., stg arthr. and stg apl. refer to stargazin from nematode, arthropod and aplysia, respectively. (B) The γ-subunit encompasses four transmembrane domains and a C-terminus that varies significantly among the different forms and that in γ₂, γ₃, γ₅ and γ₈ (shown in the inset below) ends in a TTPV motif that tethers the protein to the PDZ domains.

Figure 5

Functions of the Ras superfamily of small GTPases. (A) Subfamilies and functions of the Ras superfamily (reviewed in Vigil et al., 2010). (B) Domain structure shared by the Ras superfamily. The shared feature is the G-domain containing several GDP/GTP binding motifs (labeled G1 to G5). The Ras superfamily encompass a highly variable C-terminal domain (HVD) that includes the membrane targeting domain (MT) which undergoes lipid modification in Ras, Rho and Rab subfamilies. MT in Arf subfamily is at the N-terminus. (C) Domain organization of RGK GTPases. Note that the G domain differs from the canonical one shared by the other members of the Ras superfamily. Besides the Ca²⁺/calmodulin binding site (CaM), RGKs also contain two sites for 14-3-3 binding. The β-subunit of calcium channels binds to the G-box. (D) Models explaining RGK induced reduction of Ca²⁺ currents mediated by HVA channels (described in Yang et al., 2010). The normal function of HVAs requires the association of the β-subunit. Channel activation is preceded by the movement of the voltage sensor, represented in red. RGKs bound to GDP associate with the β-subunit and downregulate the channel by promoting its backward trafficking through a dynamin-dependent endocytosis (1) or by decreasing its open probability through a mechanism that relies on RGK membrane anchoring (2). Gating of the channel may be also altered by GTP-bound RGK through immobilization of the voltage sensor (3).

Figure 6

Structural changes of calmodulin upon binding to Ca²⁺ and IQ in Ca_V1.x and Ca_V2.x channels. The N- and C-lobes of CaM are shown in cyan and green, respectively while the linker is colored in yellow. Ca²⁺ ions are represented as red spheres and IQ as brown and violet cylinders for Ca_V1.x and Ca_V2.x, respectively. Binding of four Ca⁺² to the dumbbell-like configuration of ApoCaM (A) promotes the extended conformation of CaM with an α-helical linker (B). Binding of Ca²⁺-CaM to IQ brings the N- and C-lobes into close proximity. IQ from Ca_V1.x binds to CaM in a parallel configuration (C) and IQ-Ca_V2.x in an antiparallel manner (D). PDB files are as follows: apoCaM: 1QX5, free Ca²⁺ CaM: 1CLL, CaM with Ca_V1.2 IQ: 2BE6, CaM with Ca_V2.2 IQ 3DVE, dimer CaM with Ca_V1.2 IQ: 3G43.

Figure 7

Summary of proteins interacting with Ca_Vα₁ of HVA channels. HVA channels are heteromultimers consisting of at least three subunits (α₁, β, α₂δ), except for Ca_V1.1 class that also includes the γ-subunit. For clarity, the α₁-subunit sketch includes only the intracellular loop joining the first and second repeat (loop I–II) that associates with the β-subunit. The α₂δ- and β-subunits increase the electrical activity and the number of the channels in the plasma membrane. While the β-subunit enhances anterograde trafficking of channels likely retained in the ER, α₂δ-subunit promotes recycling of channels (curved arrow). The β-subunit also prevents the channel to be targeted to the ERAD degradation complex from the ER. RGKs regulate channel function by multiple mechanisms upon binding to the channel complex through the β-subunit (see Figure 5). CaM is widely known for its effect on HVA calcium dependent inactivation and facilitation. The exact subunit composition of the channel in the different vesicular compartments is not known. CaM, calmodulin; RGK, RGK small GTPases; RE, recycling endosome; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated protein degradation complex.