1.A.3 The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca2+ Channel (RIR-CaC) Family
Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate (IP3)-sensitive Ca2+-release channels function in the release of Ca2+ from intracellular storage sites in animal cells and thereby regulate various Ca2+-dependent physiological processes (Van Petegem 2012). They consist of (1) an N-terminal ligand binding domain, (2) a central modulatory domain and (3) a C-terminal channel-forming domain. The 3-D structure (2.2 Å) of the inositol 1,3,5-triphosphate receptor of an IP3 receptor has been solved (Bosanac et al., 2002). Structural and functional conservation of key domains in InsP(3) and ryanodine receptors has been reviewed (Seo et al., 2012). Members of the VIC (1.A.1), RIR-CaC (2.A.3) and TRP-CC (1.A.4) Families have similar transmembrane domain structures, but very different cytosolic doman structures (Mio et al. 2008).
Ry receptors occur primarily in muscle cell sarcoplasmic reticular (SR) membranes, and IP3 receptors occur primarily in brain cell endoplasmic reticular (ER) membranes where they effect release of Ca2+ into the cytoplasm upon activation (opening) of the channel. They are redox sensors, possibly providing a partial explanation for how they control cytoplasmic Ca2+. Ry receptors have been identified in heart mitochondria, and these provide the main pathway for Ca2+ entry (Beutner et al., 2001). Sun et al. (2011) have demonstrated oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel (RyR1) by NADPH oxidase 4.
The Ry receptors are activated as a result of the activity of dihydropyridine-sensitive Ca2+ channels. Ry receptors, IP3 receptors, and dihydropyridine-sensitive Ca2+ channels (TC#1.A.1.11.2) are members of the voltage-sensitive ion channel (VIC) superfamily (TC#1.A.1). Dihydropyridine-sensitive channels are present in the T-tubular systems of muscle tissues. Ry receptor 2 dysfunction leads to arrhythmias, alterred myocyte contraction during the process of EC (excitation-contraction) coupling, and sudden cardiac death (Thomas et al., 2007). Neomycin is a RyR blocker which serves as a pore plug and a competitive antagonist at a cytoplasmic Ca2+ binding site that causes allosteric inhibition (Laver et al., 2007).
Ry receptors are homotetrameric complexes with each subunit exhibiting a molecular size of over 500,000 daltons (about 5,000 amino acyl residues). They possess C-terminal domains with six putative transmembrane α-helical spanners (TMSs). Putative pore-forming sequences occur between the fifth and sixth TMSs as suggested for members of the VIC family. Recently an 8 TMS topology with four hairpin loops has been suggested (Du et al., 2002). The large N-terminal hydrophilic domains and the small C-terminal hydrophilic domains are localized to the cytoplasm. Low resolution 3-dimensional structural data are available. Mammals possess at least three isoforms which probably arose by gene duplication and divergence before divergence of the mammalian species. Homologues are present in Drosophila melanogaster and Caenorabditis elegans. Interactions of RyRs with insecticides and drugs has been reviewed (Sun and Xu 2019).
Tetrameric cardiac and skeletal muscle sarcoplasmic reticular ryanodine receptors (RyR) are large (~2.3 MDa). The complexes include signaling proteins such as 4 FKBP12 molecules, protein kinases, phosphatases, etc. They modulate the activity of and the binding of immunophilin to the channel. FKBP12 is required for normal gating as well as coupled gating between neighboring channels. PKA phosphorylation of RyR dissociates FKBP12 yielding increased Ca2+ sensitivity for activation, part of the excitation-contraction (fight or flight) response (Gaburjakova et al., 2001).
RyR1 (TC# 1.A.3.1.2) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. des Georges et al. 2016 presented cryo-EM reconstructions of RyR1 in multiple functional states, revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP, and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induces conformational changes in the cytoplasmic assembly ('priming'), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement, and deformation of the S4-S5 linker and conformational changes in the pseudo-voltage-sensor domain (des Georges et al. 2016).
IP3 receptors resemble Ry receptors in many respects (Mikoshiba, 2012). (1) They are homotetrameric complexes with each subunit exhibiting a molecular size of over 300,000 daltons (about 2,700 amino acyl residues). (2) They possess C-terminal channel domains that are homologous to those of the Ry receptors. (3) The channel domains possess six putative TMSs and a putative channel lining region between TMSs 5 and 6. (4) Both the large N-terminal domains and the smaller C-terminal tails face the cytoplasm. (5) They possess covalently linked carbohydrate on extracytoplasmic loops of the channel domains. (6) They have three currently recognized isoforms (types 1, 2, and 3) in mammals which are subject to differential regulation and have different tissue distributions. They co-localize with Orai channels (1.A.52) in pancreatic acinar cells (Lur et al., 2011).
IP3 receptors possess three domains: N-terminal IP3-binding domains, central coupling or regulatory domains and C-terminal channel domains. Channels are activated by IP3 binding, and like the Ry receptors, the activities of the IP3 receptor channels are regulated by phosphorylation of the regulatory domains, catalyzed by various protein kinases. They predominate in the endoplasmic reticular membranes of various cell types in the brain but have also been found in the plasma membranes of some nerve cells derived from a variety of tissues.
Specific residues in the putative pore helix, selectivity filter and S6 transmembrane helix of the IP3 receptor, have been mutated (Schug et al., 2008) in order to examine their effects on channel function. Mutation of 5 of 8 highly conserved residues in the pore helix/selectivity filter region inactivated the channel. Channel function was also inactivated by G2586P and F2592D mutations. These studies defined the pore-forming segment in IP (Schug et al., 2008).
The channel domains of the Ry and IP3 receptors comprise a coherent family that shows apparent structural similarities as well as sequence similarity with proteins of the VIC family (TC #1.A.1). The Ry receptors and the IP3 receptors cluster separately on the RIR-CaC family tree. They both have homologues in Drosophila. Based on the phylogenetic tree for the family, the family probably evolved in the following sequence: (1) A gene duplication event occurred that gave rise to Ry and IP3 receptors in invertebrates. (2) Vertebrates evolved from invertebrates. (3) The three isoforms of each receptor arose as a result of two distinct gene duplication events. (4) These isoforms were transmitted to mammals before divergence of the mammalian species.
In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca2+ channels and the flux of Ca2+ into the cells. This increases Ca2+ binding to ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca2+, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca2+ release events known as 'sparks.' RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Walker et al. 2016 demonstrated that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca2+ sparks. They showed that the probability of a Ca2+ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s.
Large-conductance Ca2+ release channels, ryanodine receptors (RyRs), mediate the release of Ca2+ from the endo/sarcoplasmic reticulum, to the cytoplasm. There are three mammalian RyR isoforms: RyR1 is present in skeletal muscle; RyR2 is in heart muscle; and RyR3 is expressed at low levels in many tissues including the brain, smooth muscle, and slow-twitch skeletal muscle. RyRs form large protein complexes comprising four 560-kD RyR subunits, four approximately 12-kD FK506-binding proteins, and various accessory proteins including calmodulin, protein kinases, and protein phosphatases (Meissner 2017). The greatest sequence similarity amoung RyRs is in the C-terminal region that forms the transmembrane, ion-conducting domain of ~500 aas. The remaining approximately 4,500 aas form the large regulatory cytoplasmic 'foot' structure. Experimental evidence for Ca2+, ATP, phosphorylation, and redox-sensitive sites in the cytoplasmic structure have been described. Exogenous effectors include the two Ca2+ releasing agents caffeine and ryanodine (Meissner 2017).
The generalized transport reaction catalyzed by members of the RIR-CaC family following channel activation is:
Ca2+ (out, or sequestered in the ER or SR) → Ca2+ (cell cytoplasm).
Ryanodine receptor Ca2+ release channel, RyR2. Causes Ca2+ release from the E.R. and consequent cardiac arrhythmia (Chelu and Wehrens, 2007). Associates with FKBP12.6, but phosphorylation by protein kinase A on serine-2030 causes dissociation (Jones et al., 2008). Enhanced binding of calmodulin corrects arrhythmogenic channel disorder in myocytes (Fukuda et al. 2014). RyR2s can open spontaneously, giving rise to spatially-confined Ca2+ release events known as "sparks." They are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. The spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca2+ sparks (Walker et al. 2015). Structures of RyR2 from porcine heart in both the open and closed states at near atomic resolutions have been determined using single-particle electron cryomicroscopy (Peng et al. 2016). Structural comparisons revealed breathing motions of the overall cytoplasmic region resulting from the interdomain movements of amino-terminal domains (NTDs), Helical domains, and Handle domains, whereas little intradomain shifts are observed in these armadillo repeat-containing domains. Outward rotations of the central domains, which integrate the conformational changes of the cytoplasmic region, lead to the dilation of the cytoplasmic gate through coupled motions. These observations provide insight into the gating mechanism of RyRs (Peng et al. 2016). RyR2 is subject ot regulation by cytoplasmic Zn2+ (>1nM), and this regulation plays a key role in diastolic SR Ca2+ leakage in cardiac muscle (Reilly-O'Donnell et al. 2017). Ryanodine receptor-mediated SR Ca2+ efflux is apparently balanced by concomitant counterion currents across the SR membrane (Sanchez et al. 2018).
Cardiac muscle RyR-CaC of Homo sapiens
Ryanodine receptor, RyR, of 5139 aas and 6 TMSs. Sensitive to the diamide insecticides, chlorantraniliprole and flubendiamide. It has the conserved C-terminal domain with the consensus calcium-biding EF-hands (calcium-binding motif), the six transmembrane domains, as well as mannosyltransferase, IP3R and RyR (pfam02815) (MIR) domains (Wu et al. 2018). Probably transports monovalent cations and Ca2+.
RyR of Sesamia inferens (pink stem borer)
The Ryanodine receptor Ca2+/K+ release tetrameric channel, RyR1, present in skeletal muscle, is 5038 aas long. Mutants are linked to core myopathies such as Central Core Disease, Malignant Hyperthermia and Multiple Minicore Disease) (Xu et al., 2008). RyR1 interacts with CLIC2 to modulate its channel activity (Meng et al., 2009). A model pf RyR1 has been constructed encompassing the six transmembrane helices to calculate the RyR1 pore region conductance, to analyze its structural stability, and to hypothesize the mechanism of the Ile4897 CCD-associated mutation. The calculated conductance of the wild-type RyR1 suggests that the pore structure can sustain ion currents measured in single-channel experiments. Shirvanyants et al. 2014 observed a stable pore structure with multiple cations occupying the selectivity filter and cytosolic vestibule, but not the inner chamber. Stability of the selectivity filter depends on interactions between the I4897 residue and several hydrophobic residues of the neighboring subunit. Loss of these interactions in the case of the polar substitution, I4897T, results in destabilization of the selectivity filter, a possible cause of the CCD-specific reduced Ca2+ conductance. A 4.8 Å structure of the rabbit orthologue in the closed state of this 2.3 MDa tetramer (3757 aas/protomer) reveals the pore, the VIC superfamily fold and a potential mechanism of Ca2+ gating (Zalk et al. 2015). A cryo-electron microscopy analysis revealed the structure at 6.1 Å resolution (Efremov et al. 2015). The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. They identified the calcium-binding EF-hand domain and showed that it functions as a conformational switch, allosterically gating the channel. Malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop confer RyR2-type Ca2+- and Mg2+-dependent channel regulation (Gomez et al. 2016). Structural analyses have elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism for RyR1 (Wei et al. 2016). Samsó 2016 reviewed structural determinations of RyR by cryoEM and analyzed the first near-atomic structures, revealing a complex orchestration of domains controlling channel function. The structural basis for gating and activation have been determined (des Georges et al. 2016). Junctin and triadin bind to different sites on RyR1; triadin plays an important role in ensuring rapid Ca2+ release during excitation-contraction coupling in skeletal muscle. RyR1 structure/functioin has been reviewed (Zalk and Marks 2017). Possibly, luminal Ca2+ activates RyR1 by accessing a cytosolic Ca2+ binding site in the open channel as the Ca2+ ions pass through the pore (Xu et al. 2017).
RyR1 of Homo sapiens (P21817)
The Ryanodine Receptor homologue, RyRi (5,101 aas; 77% identical to the A gambiae RyR) of the aphid, Myzus persicae, is the tartet of diamide insecticides and is made without alternative splicing (Troczka et al. 2015). The almost identical well characterized orthologue from the oriential fruit fly, Bactrocera dorsalis also has its 6 TMSs C-terminal (Yuan et al. 2014).
RyRi of Anopheles gambiae (Q7PMK5)
Ryanodine receptor (RyR) of 5107 aas. Flubendiamine, a RyR-activating insecticide, induced Ca2+ release in hemocytes (Kato et al. 2009; Wu et al. 2013).
RyR of Pieris rapae (white cabbage butterfly)
Ryanodine receptor (RyR) of 5127 aas and 6 TMSs. Intracellular calcium channel that is required for proper muscle function during embryonic development and may be essential for excitation-contraction coupling in larval body wall muscles. Mediates general anaesthesia by halothane (Gao et al. 2013) and confers sensitivity to diamide insecticides (Tao et al. 2013).
RyR of Drosophila melanogaster (Fruit fly)
Ryanodine-sensitive calcium release channel receptor, RyR of 5071 aas and 6 putative TMSs. The tissue lecalization has been described (Hamada et al. 2002). Required for neuronal regeneration (Sun et al. 2014).
RyR of Caenorhabditis elegans
Aphid ryanodine receptor RyR) of 5105 aas and 6 TMSs, a target of insecticides. The sequence of the Acyrthosiphon pisum (Pea aphid) is provided below, but the Toxoptera citricida (98% identiy; Brown citrus aphid; Aphis citricidus) RyR was studied (Wang et al. 2015).
Aphid RyR of Acyrthosiphon pisum (Pea aphid)
Ryanodine receptor, DcRyR shows high sequence identity to RyRs from other insects (76%-95%) and shares many features of insect and vertebrate RyRs, including a MIR domain, two RIH domains, three SPRY domains, four copies of RyR repeat domain, an RIH-associated domain at the N-terminus, two consensus calcium-binding EF-hands and six TMSs at the C-terminus (Yuan et al. 2017). The expression of DcRyR mRNA was the highest in the nymphs and lowest in eggs; it has three alternative splice sites, and the splice variants showed body part-specific expression, being under developmentally regulation (Yuan et al. 2017).
RyR of Dialeurodes citri (Citrus whitefly) (Aleurodes citri)
Ryanodine-sensitive Ca2+ release channel RyR1 of 5117 aas and 6 TMSs. Diamide insecticides, such as flubendiamide and chlorantraniliprole, selectively activate insect ryanodine receptors. They are particularly active against lepidopteran pests of cruciferous vegetable crops, including the diamondback moth, Plutella xylostella. Resistance results from mutation(s) in the ryanodine receptors' transmembrane domain (Troczka et al. 2017).
RyR1 of Plutella xylostella (Diamondback moth) (Plutella maculipennis)
Inositol 1,4,5-trisphosphate receptor-2 with 2701 aas and 6 TMSs. Mediates release of intracellular calcium which is regulated by cAMP both dependently and independently of PKA and plays a critical role in cell cycle regulation and cell proliferation. High level expression in humans is an indication of cytogenetically normal acute myeloid leukemia (CN-AML) (Shi et al. 2015).
Brain IP3-CaC of Rattus norvegicus
Calcium release channel III, CRCIII1a of 2598 aas. Associated with recycling vesicles engaged in phagosome formation (Ladenburger and Plattner 2011).
CRCIII1a of Paramecium tetraurelia
Calcium release channel IV3b, CRCIV3b, of 3127 aas. Display structural and functional properties of ryanodine receptors (Ladenburger et al. 2009). Localized to the alveolar sacs of the cortical subplasmalemmal Ca2+-stores (Plattner et al. 2012). Involved in exocytosis in response to ryanodine receptor agonists (Docampo et al. 2013).
CRCIV3b of Paramecium tetraurelia
Calcium release channel V-4b, CRCV4b of 2589 aas. Occurs in parasomal (alveolar) sacs (clathrin coated pits) (Docampo et al. 2013).
CRCV4b of Paramecium tetraurelia
Calcium release channel VI-2b, CRCVI2b. Localized to the contractile vacuole (Docampo et al. 2013).
CRCVI2b of Paramecium tetraurelia
Endoplasmic reticular inositol triphosphate receptor, IP3R of 3099 aas (Docampo et al. 2013).
IP3R of Trypanosoma brucei
Inositol triphosphate receptor, IP3R, also called Itr-1, Dec-4 and Ife-1, of 2892 aas and 6 TMSs (Baylis and Vázquez-Manrique 2012). Receptor for inositol 1,4,5-trisphosphate, a second messenger that regulates intracellular calcium homeostasis. Binds in vitro to both 1,4,5-InsP3 and 2,4,5-InsP3 with high affinity and does not discriminate between the phosphate at the 1 or 2 position. Can also bind inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-InsP4) and inositol 4,5-bisphosphate (4,5-InsP2), but with lower affinity. Acts as a timekeeper/rhythm generator via calcium signaling, affecting the defecation cycle and pharyngeal pumping (Dal Santo et al. 1999). Affects normal hermaphrodite and male fertility as a participant in intracellular signaling by acting downstream of let-23/lin-3 which regulates ovulation, spermathecal valve dilation and male mating behavior (Walker et al. 2002; Gower et al. 2005). Important for early embryonic development; controls epidermal cell migration and may also regulate filopodial protrusive activity during epithelial morphogenesis (Thomas-Virnig et al. 2004; ). Component of inositol trisphosphate (IP3)-mediated downstream signaling pathways that controls amphid sensory neuronal (ASH)-mediated response to nose touch and benzaldehyde (Walker et al. 2009).
IP3 receptor of Caenorhabditis elegans
IP3R of 3140 aas, RyR1 (Wheeler and Brownlee 2008).
IP3R of Chlamydomonas reinhardtii
The Inositol 1,4,5- triphosphate (InsP3)-like receptor (2838aas). Receptor for inositol 1,4,5-trisphosphate, a
second messenger that mediates the release of intracellular calcium.
May be involved in visual and olfactory transduction as well as myoblast
proliferation. Loss in adult neurons results in obesity in adult flies (Subramanian et al. 2013).
InsP3l receptor Drosophila melanogaster (P29993)
InsP3-like protein of Tetrahymena themophila (Q23K98)
The Inositol 1,4,5- triphosphate (InsP3)-like receptor (3036aas) (Ladenburger et al. 2009; Docampo et al. 2013).
InsP3l receptor of Paramecium tetraaurelia (A0CX44)
Inositol 1,4,5-triphosphate receptor type 1 splice variant, IP(3)R1 (Subedi et al., 2012). The human orthologue, IP3R3, is regulated at the ER-mitochondrion interface by BCL-XL (TC# 1.A.21.1.6) (Williams et al. 2016).
IP(3)R1 of Rattus norvegicus (Q63269)
Contractile vacuole complex calcium release channel (CRC)II; IP3Rn (Ladenburger et al. 2006). Functions in osmoregulation by promoting expulsion of water and some ions including Ca2+. Also functions in calcium homeostasis (Ladenburger et al. 2006; Docampo et al. 2013).
IP3Rn or CRCII of Paramecium trtraurelia
Putative IP3R calcium-release channel VI-3 of 2021 aas (Docampo et al., 2013).
Calcium-release channel VI-3 of Paramecium tetraurelia
CRCI-1a; IP3R. Functions similarly to TC# 1.A.3.2.7 (Docampo et al. 2013). Cortical Ca2+ stores (alveolar sacs) are activated during stimulated trichocyst exocytosis, mediating store-operated Ca2+ entry (SOCE). Ca2+ release channels (CRCs) localise to alveoli and are Ryanodine receptor-like proteins (RyR-LPs) as well as inositol 1,4,5-trisphosphate receptors (IP3Rs), members of the CRC family with 6 subfamilies (Plattner 2014).
CRCI-1a of Paramecium tetraurelia