8.A.16 The Ca⁺ Channel Auxiliary Subunit γ1-γ8 (CCAγ) Family

Calcium channel γ1-8 auxiliary subunits all share limited sequence similarity and belong to a single family. They have the same topology of four transmembrane segments (a claudin domain) including a large loop between TMSs 1 and 2, a characteristic of TC family 1.H.2. All neuronal γ-subunits share a C-terminal consensus site for phosphorylation by cAMP/cGMP-dependent protein kinases. They associate with α-subunits of voltage-gated Ca²⁺ channels (TC #1.A.1.11). Phylogenetic, bioinformatic, and functional studies indicated that these proteins are functionally diverse. A cluster containing gamma1 and gamma6 act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (gamma2, gamma3, gamma4, gamma8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (gamma5, gamma7) remains unclear. Chen et al. 2007 showed that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. Binding of TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions (Ben-Yaacov et al. 2017).

γ2 (also called stargazin) and γ3 have been shown to associate with P/Q- and N-type channels.  Regulation by stargazin may occur via the G-beta-gamma subunits of Ca(v)2.2 (TC# 1.A.1.11.19) and G protein-activated inward rectifier potassium channels, GIRK (see 1.A.2.1.3; Tselnicker and Dascal 2010). γ3 and γ4 are express only in neuronal tissues. γ1 and γ6 have two glycosylation sites, and this may be a characteristic of all γ-subunits. Binding of a TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions (Ben-Yaacov et al. 2017).

The eight members of the calcium channel gamma subunit family regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ2, 3, 4 and 8,  (the TARPs) modulate AMPA receptor localization and function, the γ1 and 6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels.  γ6 mediates inhibition of the low voltage activated Cav3.1 channel by direct interaction involved the GxxxA motif in TMS1 (Lin et al. 2008). Modulation of TARP gamma8-containing AMPA receptors provides a therapeutic Aapproach for chronic pain (Knopp et al. 2019).

These putative calcium channel auxiliary subunits are found in eukaryotic genomes including human, rat, mouse, fugu fish, chicken, zebra fish, fruitfly, and C. elegans. This family may function in the assembly, modulation of function and/or maintenance of structure of skeletal muscle and brain calcium channels. γ1 controls the dihydropyridine-sensitive L-type skeletal muscle calcium channel (TC #1.A.1.11.2). The determinants of AMPAR regulation by TARPs has been reviewed, showing why TARPs display a broad range of effects despite their conserved modular architecture (Twomey et al. 2019).

Transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor (TC# 1.A.10) regulatory proteins (TARPs) are auxiliary subunits that regulate AMPA receptor trafficking to the plasma membrane, localization to postsynaptic sites and channel gating (Nicoll et al. 2006). The classical TARP family consists of four members: stargazin/gamma-2, gamma-3, gamma-4 and gamma-8. The TARP gamma-8 isoform, which is highly expressed in the hippocampus, has a unique, long, C-terminal domain with five distinct regions: two glycine- rich regions, a serine/arginine-rich region, a proline/alanine (P/A)-rich region, and a PSD-95/Dlg/ZO-1 (PDZ) binding motif. Itakura et al. 2014 performed mass spectrometry and immunoprecipitation assays to identify specific binding partners for the gamma-8 C-terminal tail and found that gamma-8, but not stargazin/gamma-2, co-immunoprecipitated with calcineurin/PP2B, a Ca²⁺/calmodulin-dependent Ser/Thr phosphatase. Co-immunoprecipitation and immunoblot analyses of lysates from COS-7 cells co- transfected with calcineurin and either wild type or chimeric gamma-8 revealed that a section of the C-terminal tail (residues 356-421) can bind calcineurin. Futhermore, gamma-8 lacking the P/A-rich region (residues 383-399) did not bind to calcineurin. In addition, the GST-gamma-8 C-terminal tail (residues 353-414) fusion protein containing the P/A-rich region bound to purified calcineurin in a Ca²⁺/calmodulin-dependent manner, whereas GST-gamma-8 with a deletion of the P/A-rich region did not. Peptide competition assays demonstrated that gamma-8 may interact with the hydrophobic pocket defined by beta-sheet 14 and/or adjacent regions of the catalytic A subunit of calcineurin. Thus, the gamma-8 P/A-rich region is essential for binding calcineurin, suggesting that the gamma-8/calcineurin complex may regulate AMPA receptor phosphorylation and trafficking (Itakura et al. 2014).

Transmembrane AMPA receptor regulatory proteins (TARPs) govern AMPA receptor cell surface expression and distinct physiological properties including agonist affinity, desensitization and deactivation kinetics. The prototypical TARP, STG or gamma2 and TARPs gamma3, gamma4, gamma7 and gamma8 are all expressed to varying degrees in the mammalian brain and differentially regulate AMPAR gating parameters. Positive allosteric AMPA receptor modulators or ampakines alter receptor rates of agonist binding/unbinding, channel opening and can offset receptor desensitization and deactivation. The effects of the two ampakines, CX614 and cyclothiazide (CTZ) have been evaluated on homomeric GluR1-flip receptors and GluR2-flop receptors with or without different TARPs, gamma2, gamma3, gamma4 or gamma8 genes (Radin et al. 2018). gamma4 was the most robust TARP in increasing the affinities of CX614 and CTZ on GluR1-flip receptors, but had no such effect on GluR2-flop receptors. However, gamma8 gave the most significant increases in affinities of CX614 and CTZ on GluR2-flop. Thus, TARPs differentially affect the surface expression and kinetics of the AMPA receptor, as well as the pharmacology of ampakines for the AMPA receptor. The modulatory effects of TARPs on ampakine pharmacology are complex, being dependent on both the TARP subtype and the AMPA receptor subtypes/isoforms (Radin et al. 2018).