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).
Calcium channel γ1-subunit, CACNG1 of 222 aas and 4 TMSs. Evolution of the family of gamma subunits has been presented and discussed (Burgess et al. 2001). Mutations in genes encoding proteins involved in ion channel function (including CACNG1) may be responsible for the aggressive behavior of a tumor (Sharma et al. 2019).
CACNG1 from Homo sapiens
Calcium channel γ6-subunit, CACNG6. Mediates inhibition of the low voltage-activated Cav3.1 channel by direct interaction involving a GxxxA motif in TMS1 (Lin et al. 2008).
CACNG6 from Homo sapiens
Transmembrane AMPA receptor (AMPAR) regulatory proteins, also called voltage-dependent Ca2+ channel γ2 subunits, TARPγ2, CACNG2, gamma-2 or Stargazin; regulate Ca(v)2.2 (TC# 1.A.1.11.19) as well as GIRK (TC# 1.A.2.1.3) (Tselnicker and Dascal 2010). TARPγ2 and TARPγ7 (8.A.16.2.5) can substitute for each other (Yamazaki et al. 2015). TARPs also enhance AMPAR function, altering ligand efficacy and receptor gating kinetics and shaping the postsynaptic response. Stargazin rescues gating deficiencies in AMPARs carrying mutations that destabilize the closed-cleft states of the ligand-binding domain (LBD), suggesting that stargazin stabilizes closed LBD states (MacLean et al. 2014). For Ca2+-permeable AMPARs, stargazin enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines (McGee et al. 2015). TARPs alter the conformations of pore-forming subunits and thereby affect antagonist interactions (Cokić and Stein 2008). By shifting the balance between AMPAR activation and desensitization, TARPs markedly facilitate the transduction of spillover-mediated synaptic signaling (Coombs et al. 2017). Stargazin plays a role in AMPA receptor trafficking through the early compartments of the biosynthetic pathway (Vandenberghe et al. 2005). Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping (Zeng et al. 2019). Positive allosteric modulators (PAMs) of AMPA receptors boost cognitive performance in clinical studies, and mibampator and BIIB104 discriminate between AMPARs complexed with distinct TARPs, and particularly those with lower stargazin/gamma2 efficacy such as BIIB104 (Ishii et al. 2020). TARPs modulate receptor-mediated paired-pulse depression and recovery from desensitization (Devi et al. 2020). The influence of the TARP gamma8-selective negative allosteric modulator JNJ-55511118 on AMPA receptor gating and channel conductance has been studied (Coombs et al. 2022). Aberrant hippocampal transmission and behavior in mice with a stargazin mutation has been linked to intellectual disability (Caldeira et al. 2022).
CACNG2 of Homo sapiens (Q9Y698)
Voltage-dependent calcium channel gamma-8 subunit, a duplicated 8 TMS protein of 602 aas with two 4 TMS elements, with the first half belonging to subfamily 8.A.16.2 and the second half more similar to subfamily 8.A.16.1.
Voltage-dependent calcium channel gamma-8 subunit of Tupaia chinensis (Chinese tree shrew)
Voltage-dependent calcium channel gamma-6 subunit, a duplicated 8 TMS protein of 630 aas with two 4 TMS elements, the first half belonging to subfamily 8.A.16.2 and the second half more similar to subfamily 8.A.16.1. This protein and 8.A.16.2.10 have internal duplications and 8 TMSs instead of the usual 4 TMSs.
Voltage-dependent calcium channel gamma-6 subunit of Heterocephalus glaber (Naked mole rat)
TARP Cacng2a (Stargazin homologue) of 324 aas. Regulates the trafficking and gating properties of AMPA-selective glutamate receptors (AMPARs), promoting their targeting to the cell membrane and synapses and modulating their gating properties by slowing their rates of activation, deactivation and desensitization. Regulates all AMPAR subunits. Thought to stabilize the calcium channel in an inactivated (closed) state (Roy et al. 2016).
Cacng2a of Danio rerio (Zebrafish) (Brachydanio rerio)
CACNG3 of Homo sapiens (O60359)
Voltage dependent Ca2+ channel γ5 subunit, CACNG5
CACNG5 of Mus musculus (Q8VHW4)
Voltage dependent Ca2+ channel γ7 (γ-7) subunit, CACNG7 or TARP γ7. Enhances synaptic expression and channel activity of Ca2+ permeable AMPA receptors (TC#1.A.10.1.1) (Studniarczyk et al. 2013; Kato et al. 2007).
CACNG7 of Homo sapiens (P62955)
Voltage dependent Ca2+ channel γ8 subunit, CACNG8 or TARP γ8, of 425 aas and 4 or 5 TMSs, interacts with calcineurin to regulate AMPA receptor phosphorylation and trafficking (Itakura et al. 2014). It regulates the activity of L-type calcium channels that contain CACNA1C as the pore-forming subunit (Shi et al. 2010). It also regulates the gating properties of AMPARs while promoting their targeting to the cell membrane and synapses and modulating their gating properties by slowing their rates of activation, deactivation and desensitization and by mediating their resensitization (Kato et al. 2010). It does not show subunit-specific AMPA receptor regulation and regulates all AMPAR subunits. TARP gamma8 is predominantly expressed in the forebrain and is enriched in the hippocampus, a region associated with temporal lobe epilepsy, and may allow development of refined AMPAR epilepsy therapeutics (Dohrke et al. 2020). SNP rs10420324 in the AMPA receptor auxiliary subunit TARP gamma-8 regulates the susceptibility to antisocial personality disorder (Peng et al. 2021). The TARP gamma8-selective negative allosteric modulator JNJ-55511118 influences AMPA receptor gating and channel conductance (Coombs et al. 2022). TARP gamma-8 regulates the AMPAR which mediates various brain functions including learning and memory, and it has been imaged (Yu et al. 2022). A deficiency of transmembrane AMPA receptor regulatory protein gamma-8 leads to attention-deficit hyperactivity disorder-like behavior in mice (Bai et al. 2022).
CACNG8 of Homo sapiens (Q8WXS5)
The cataract-associated protein TMEM114 (222 aas) (Glycosylated) (Maher et al., 2011)
TMEM114 of Mus musculus (Q9D563)
Tetrameric tetraspanin MP20 (4 TMS scaffold protein; Gonen et al., 2007). Proteomics of the ocular lens revealed palmitoylation (Wang and Schey 2018).
Tetraspanin MP20 of Rattus norvegicus (P54825)
Uncharacterized protein of 273 aas and 4 TMSs. According to Pfam, it belongs to the Claudin2 superfamily.
TMEM202 homologue of Homo sapiens (Human)
Invertebrate claudin-like cell junctional protein, Vab-1, of 211 aas and 4 TMSs. Regulates cell adhesion, intercellular signalling, cell morphology and paracellular small molecule passage (Simske and Hardin 2011).
Vab-1 of Caenorhabditis elegans
Uncharacterized protein of 198 aas and 4 TMSs
UP of Capitella teleta (Polychaete worm)
p53 apoptosis effector, related to PMP-22, PERP, PCP1, KRTCAP1, PIGPC1, THW, of 193 aas and 4 TMSs in a 1 + 3 TMS arrangement (Attardi et al. 2000).
PERP of Homo sapiens
TMEM47; BCMP1; TM4SF10 of 181 aas and 4 TMSs. It regulates cell junction organization in epithelial cells and may play a role in the transition from adherens junction to tight junction assembly. It is a possible biomarkers for accurate identification of mesenchymal stromal cells (Brinkhof et al. 2020)
TMEM47 of Homo sapiens
Germ cell-specific gene 1 protein of 349 aas and 4 TMSs, GSG1.
GSG1 of Homo sapiens
Uncharacterized protein of 245 aas and 4 TMSs.
UP of Branchiostoma floridae (Florida lancelet)
Germ cell-specific gene-1-like (GSG1L) protein of 331 aas and 4 TMS in a 1 (N-terminal) + 3 TMS arrangement. Calcium-permeable AMPA receptors (TC# 1.A.10) are specific for the neurotransmitter glutamate. They contribute to various forms of synaptic plasticity. Alterations in their expression or regulation are seen in a number of neurological conditions including stroke, motor neuron disease, and cocaine addiction. Several groups of auxiliary transmembrane proteins have been described that enhance the function and cell-surface expression of AMPARs, but GSG1L decreases channel conductance and calcium permeability while increasing polyamine-dependent rectification by diminishing outward current (McGee et al. 2015). GSG1L favors the AMPAR desensitized state, where channel closure is facilitated by large structural rearrangements in the AMPAR extracellular domain, with ligand-binding domain dimers losing their local 2-fold rotational symmetry. AMPAR auxiliary subunits probably share a modular architecture (Twomey et al. 2017). It may also be a subunit of AMPA receptors (Matthews et al. 2021).
GSG1L OF Homo sapiens