2.A.19 The Ca2+:Cation Antiporter (CaCA) Family
Proteins of the CaCA family are found ubiquitously, having been identified in animals, plants, yeast, archaea and divergent bacteria. They exhibit widely divergent sequences, and several have been shown to have arisen by a tandem intragenic duplication event (Saier et al., 1999). The most conserved portions of this repeat element, α1 and α2, are found in putative TMSs 2-3 and TMSs 7-8 in the model of Iwamoto et al. (1999). These sequences are important for transport function and may form an intramembranous pore/loop-like structure. These carriers function primarily in Ca2+ extrusion (DiPolo and Beauge, 2006). The human Na+:Ca2+ exchangers, when defective, can cause neurodegenerative disorders (Gomez-Villafuertes et al., 2007). Allosteric activation of NCX involves the binding of cytosolic Ca2+ to regulatory domains CBD1 and
CBD2 (Giladi and Khananshvili 2013).
The CaCA superfamily is composed of five families: K+-independent Na+/Ca2+ exchangers (NCXs), cation/Ca2+ exchangers (CCXs), YbrG transporters and cation exchangers (CAXs). Phylogenetic and alignment studies indicate that one of the mammalian NCKXs, NCKX6 (2.A.19.4.4), and its related proteins form a unique group, designated cation/Ca2+ exchangers (CCXs) (Cai and Lytton, 2004). Cytoplasmic Ca2+ regulates dimeric Na+/Ca2+ exchangers (NCX) by binding to two adjacent Ca2+-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6, and produces structural rearrangements (John et al., 2011). All NCX proteins encoded in the genomes of rice and Arabidopsis have been studied with respect to their phylogeny, domain architecture and expression profiles across different tissues, at various developmental stages and under stress conditions (Singh et al. 2015).
Members of the CaCA family vary in size from 302 amino acyl residues (Methanococcus jannaschii) to 1199 residues (Bos taurus). Even within the animal kingdom, they vary in size from 461 to 1199 residues. The bacterial and archaeal proteins are in general smaller than the eukaryotic proteins (Chung et al., 2001). They have been suggested to traverse the membrane 9 (mammals) or 10 (bacteria) times as α-helical spanners, but some plant homologues (Cax1 and Cax2) exhibit 11 putative TMSs. The E. coli ChaB (YrbG) homologue has been found to have 10 TMSs with both the N- and C-termini localized to the periplasm. Each homologous half of the internally duplicated protein has 5 TMSs with opposite orientation in the membrane (Saaf et al., 2001). This orientation seems to be stabilized by the presence of positively charged residues in the cytoplasmic loops.
The mammalian cardiac muscle homologue probably has 9 TMSs. The N-terminus of this protein is believed to be extracellular, while the C-terminus is intracellular (Iwamoto et al., 1999). A large central loop is not required for transport function and plays a role in regulation. In the preferred 9 TMS model for this mammalian protein, the polypeptide chain loops into the membrane after TMS 2 and after TMS 7. The large central loop separates TMS 5 from TMS 6. TMS 2 and the following loop show sequence similarity to TMS 7 and its loop. TMS 7 may be close to TMSs 2 and 3 in the 3-D structure of the protein (Qui et al., 2001).
The Na+:Ca2+ exchanger plays a central role in cardiac contractility by maintaining Ca2+ homeostasis. Two Ca2+-binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca2+ binding to these regulatory domains activates the transport of Ca2+ across the plasma membrane. The structure of CBD1 shows four Ca2+ ions arranged in a tight planar cluster. The structure of CBD2 in the Ca2+-bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations shows (like CBD1) a classical Ig fold but coordinates only two Ca2+ ions in primary and secondary Ca2+ sites. In the absence of Ca2+, Lys585 stabilizes the structure by coordinating two acidic residues (Asp552 and Glu648), one from each of the Ca2+-binding sites, and prevents protein unfolding (Besserer et al., 2007).
All of the characterized animal proteins catalyze Ca2+:Na+ exchange although some also transport K+. The NCX plasma membrane proteins exchange 3 Na+ for 1 Ca2+ (i.e., 2.A.19.3). Mammalian Na2+/Ca2+ exchangers exist as three isoforms NCX1-3 which are about 70% identical to each other. The NCKX exchangers exchange 1 Ca2+ plus 1 K+ for four Na+ (i.e., 2.A.19.4). The myocyte NCX1.1 splice variant catalyzes Ca2+ extrusion during cardiac relaxation and may catalyze Ca2+ influx during contraction. The E. coli ChaA protein catalyzes Ca2+:H+ antiport but may also catalyze Na+:H+ antiport slowly. All remaining well-characterized members of the family catalyze Ca2+:H+ exchange.
The Na+/Ca2+ exchanger, NCX1 (TC #2.A.19.3.1), is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is regulated by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The high-affnity binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. A crystal structure of CBD1 at 2.5Å resolution reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing Nicoll et al., 2006).
The phylogenetic tree for the CaCA family reveals at least six major branches (Saier et al., 1999). Two clusters consist exclusively of animal proteins, a third contains several bacterial and archaeal proteins, a fourth possesses yeast, plant and blue green bacterial homologues, the fifth contains only the ChaA Ca2+:H+ antiporter of E. coli and the sixth contains only one distant S. cerevisiae homologue of unknown function. Several homologues may be present in a single organism. This fact and the shape of the tree suggest either that isoforms of these proteins arose by gene duplication before the three domains of life split off from each other or that horizontal gene transfer has occurred between these domains (Saier et al., 1999).
Prokaryotic CaCAs contain only the transmembrane domain and is self-sufficient as an active ion transporter, but the eukaryotic NCX proteins possesses in addition, a large intracellular loop that senses intracellular calcium signals and controls the activation of ion transport across the membrane. This provides a necessary layer of regulation for the more complex function. The Ca2+ sensor in the intracellular loop (the Ca2+-binding domain (CBD12)) signals, and allosteric intracellular Ca2+ binding propagates a signal. Another structured domain that is N-terminal to CBD12 in the intracellular loop has two tandem long α-helices, connected by a short linker. It forms a stable cross-over two-helix bundle, resembling an 'awareness ribbon' (Yuan et al. 2018).
Homologues from several cyanobacteria play important roles in salt tolerance (Waditee et al., 2004).
The generalized transport reaction catalyzed by proteins of the CaCA family is:
Ca2+ (in) + [nH+ or nNa+ (out)] ⇌ Ca2+ (out) + [nH+ or nNa+] (in)
This family belongs to the Cation Diffusion Facilitator (CDF) Superfamily.
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|2.A.19.1.1||Ca2+:H+ antiporter (also catalyzes Na+:H+ and K+:H+ antiport in processes that have been shown to be physiologically important under certain conditions) (Ivey et al., 1993; Radchenko et al., 2006)||Bacteria ||ChaA of E. coli |
Putative CaCA family member of 368 aas and 10 TMSs
Amoebozoa (Slime molds)
UP of Dictyostelium discoideum
Uncharacterized protein of 518 aas and 10 TMSs
UP of Trichoplax adhaerens (Trichoplax reptans)
|2.A.19.2.1||Ca2+:H+ antiporter ||Bacteria ||Ca2+:H+ antiporter of Synechocystis |
|2.A.19.2.10||Vacuolar cation/proton exchanger 1a (Ca(2+)/H(+) exchanger 1a) (OsCAX1a)||Plants||CAX1a of Oryza sativa subsp. japonica |
The Ca2+:H+ antiporter, YfkE; homotrimer with subunit size of 451 aas. The 3-d x-ray strcuture is known to 3.1 Å resolution (Wu et al. 2013). The conformational
transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is
mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. The inward facing conformation contrasts with the outward facing conformation demonstrated for NCX_Mj (TC# 2.A.19.5.3). The inward facing conformation has a "hydrophobic seal" that closes the external exit.
YfkE of Bacillus subtilis
Vacuolar [Mn2+ or Ca2+]:H+ antiporter, Hum1 (Mn2+ resistance (Mnr1)) protein. Vcx1 has 11 probable TMSs with the N-terminus inside (Segarra and Thomas, 2008). The 3-d structure has been determined at 2.3 Å resolution for the cytosolic facing, substrate bound form, favoring the alternating access mechanism of transport (Waight et al. 2013).
Hum1 (Mnr1) of Saccharomyces cerevisiae
|2.A.19.2.3||High affinity vacuolar (tonoplast) Ca2+:H+ antiporter (also exports Cd2+ and Zn2+; Shigaki et al., 2005) expressed in leaves (Cheng et al., 2005). (Determines sensitivity to abscisic acid and sugar during germination and tolerance to ethylene during early seedling development (Zhao et al., 2008))||Plants ||Cax1 of Arabidopsis thaliana|
Low affinity Ca2+:H+/heavy metal cation (e.g., Mn2+, Mg2+, Cd2+, Ca2+):H+ antiporter, Cax2
Cax2 of Arabidopsis thaliana
|2.A.19.2.5||High affinity vacuolar (tonoplast) Ca2+:H+ antiporter (also exports Cd2+ and Zn2+; Shigaki et al., 2005) highly expressed in roots (Cheng et al., 2005) (exhibits phenotypes characteristic of CAX1, but also determines sensitivities to salt, lithium and low pH (Zhao et al., 2008)||Plants ||Cax3 of Arabidopsis thaliana
Algae Ca2+: H+ and Na+:H+ exchanger, CAX1 (mediates stress responses to high Ca2+, Na+ and Co2+).
CAX1 of Chlamydomonas reinhardtii (B6ZCF4)
|2.A.19.2.7||Ca2+/H+ antiporter, YfkE (Fujisawa et al., 2009).|
YfkE of Bacillus subtilis (O34840)
The vacuolar Ca2+:H+ exchanger, CAX (Bowman et al., 2011).
CAX of Neurospora crassa (O59940)
Vacuolar cation:proton exchanger, Cax4 (transports Cd2+>Zn2+>Ca2+>Mn2+) (Cheng et al., 2002; Mei et al., 2009). The rice orthologue, Cax4, may transport Ca2+, Mn2+ and Cu2+, and functions in salt stress (Yamada et al. 2014).
Cax4 of Arabidopsis thaliana (Q945S5)
The 10 TMS cardiac Ca2+:3 Na+ antiporter, NCX1 (Ren and Philipson 2013). The Ca2+ sensor (residues 371-508) binds cytoplasmic Ca2+ allosterically to activate exchange activity) (Nicoll et al., 2006; Ren et al., 2006) NCX1 forms homodimers (Ren et al., 2008). It is present in mitochondria where it catalyzes Ca2+ efflux. TMS packing has been analyzed by Ren et al. (2010). Cytoplasmic Ca2+ regulates the dimeric NCX by binding to two adjacent Ca2+-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. John et al. (2011) showed that Ca2+decreases the distance between the cytoplasmic loops of NCX pairs, thereby activating transport. Ser110 in TMS2 plays a role in both Na+ and Ca2+ transport (Ottolia and Philipson 2013). nimodipiine-sensitive NCX1, as well as mitochondrial Ca2+ uptake, plays an important role in clearing somatic Ca2+ after depolarization-induced Ca2+ influx in SCN neurons (Wang et al. 2015). Regulation in suprachiasmatic nucleus neurons has been studied (Cheng et al. 2018).
suprachiasmatic nucleus neurons
Ca2+ regulated Ca2+:Na+ antiporter (NCX1) of Bos taurus
Probable Ca2+:3Na+ antiporter, Calx (contains two repeat motifs Calx-α and Calxβ, between the two transmembrane domains, as is true of many Ca2+:Na+ antiporters (Schwarz and Benzer, 1997). CALX activity is inhibited by Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2. The Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1 (Wu et al., 2011).
Calx of Drosophila melanogaster
Plasma membrane sodium:calcium exchanger, NCX3, NAC3 or SLC8A3, controlling Ca2+ homeostasis. Extrudes 1 Ca2+ for 3 extracellular Na+ ions. Potent inhibitors have been identified (Secondo et al. 2015). One such inhibitor of NCX transporters, ORM-10962, exhibits high efficacy and selectivity. Selective NCX inhibition can exert positive as well as negative inotropic effects, depending on the actual operation mode of the NCX (Kohajda et al. 2016).
SLC8A3 of Homo sapiens
Sodium/calcium exchanger 1 (Na+/Ca2+-exchange protein 1). The cardiac isoform, CAX1.1, like the archaeal homologues for which high resolution 3-d structures are available (TC#s 2.A.19.5.3 and 2.A.19.8.2), have two aqueous ion permeation channels with cavities that can face the cytoplasm or the external medium (John et al. 2013). It exchanges one Ca2+ ion against three to four Na+ ions, and thereby contributes to the regulation of cytoplasmic Ca2+ levels and Ca2+-dependent cellular processes (Komuro et al. 1992; , Van Eylen et al. 2001; Kofuji et al. 1992). It also contributes to Ca2+ transport during excitation-contraction coupling in muscle. In a first
phase, voltage-gated channels mediate the rapid increase of cytoplasmic
Ca2+ levels due to release of Ca2+ stores from the endoplasmic reticulum. SLC8A1 mediates the export of Ca2+ from the cell during the next phase, so that cytoplasmic Ca2+ levels rapidly return to baseline. It is also required for normal embryonic heart development and the onset of heart contractions. Both NCX1 and NCX2 play important roles in the motility of the gastric fundus, ileum and
distal colon (Nishiyama et al. 2016). An amphipathic α-helix in the NCX1 large intracellular loop controls NCX1 palmitoylation. Thus, NCX1 palmitoylation is governed by a distal secondary structure element rather than by local primary sequence (Plain et al. 2017).
SLC8A1 of Homo sapiens
Sodium/calcium exchanger 2 (Na+/Ca2+-exchange protein 2; NCX2; SLC8A2) of 921 aas and 11 TMSs. Functional inhibition of NCX2 initially causes natriuresis, and further
inhibition produces hypercalciuria, suggesting that the functional significance of NCX2 lies
in Na+ and Ca+ reabsorption in the kidney (Gotoh et al. 2015). However NCX1-3 are present in the brain where they influence stroke theraputic strategies in a NCX subtype-specific fashion (Shenoda 2015). NCX1 and NCX2 play important roles in the motility of the gastric fundus, ileum and
distal colon, but only NCX2 plays a role in the development of diarrhea (Nishiyama et al. 2016).
SLC8A2 of Homo sapiens
Full length G-protein coupled receptor 98 (GPR98); Also called Monogenic audiogenic seizure susceptibility protein 1 homologue; Usher syndrome type-2C protein; Very large G-protein coupled receptor 1 (VLGR1). Plays a role in CNS development; exists as multiple processed isoforms.
GPR98 of Homo sapiens
|2.A.19.4.1||Rod photoreceptor Ca2+ + K+:4 Na+ antiporter, NCKX1 || Animals ||Ca2+ + K+:Na+ antiporter (NCKX1) of Bos taurus |
Sodium/potassium/calcium exchanger 3 (Na+/K+/Ca2+-exchange protein 3), NCKX3, or (Solute carrier family 24 member 3), SLC24A3 (Yang et al. 2013).
SLC24A3 of Homo sapiens
|2.A.19.4.11||Sodium/potassium/calcium exchanger 2 (Na(+)/K(+)/Ca(2+)-exchange protein 2) (Retinal cone Na-Ca+K exchanger) (Solute carrier family 24 member 2)||Animals||SLC24A2 of Homo sapiens|
Putative Ca2+:cation exchanger of 1524 aas and an apparent duplication with 27 putative
TMSs and at leaswt 4 repeat units of 4 - 6 TMSs in the arrangement: 1 - 4, 1 - 220 aas; 5 - 9, 240 - 410 aas; 10 - 15, 460 - 650 aas; 16 - 21, 660 - 940 aas; and 22 - 27, 990 - 1200 aas. It also has a C-terminal PAN-APP domain as in plasminogen.
Putative Ca2+: cation exchanger of Branchiostoma floridae
Uncharacterized protein of 623 aas.
UP of Aureococcus anophagefferens (Harmful bloom alga)
Ca2+:Na+ exchanger, NCX-9 of 651 aas and 14 TMSs. Plays a role in developmental cell patterning and Ca2+ exchange in mitochondrial (Sharma et al. 2017).
NCX-9 of Caenorhabditis elegans
|2.A.19.4.2||The major neuronal Ca2+ + K+:4 Na+ antiporter, NCKX2||Animals||NCKX2 of Rattus norvegicus |
|2.A.19.4.3||The sea urchin spermatozoan flagellar K+-dependent Ca2+:Na+ antiporter SuNCKX (Ca2+ + K+:4 Na+ antiporter) ||Animals||SuNCKX of Strongylocentrotus purpuratus|
K+-dependent Na+/Ca2+ antiporter, NCKX6 (Cai and Lytton, 2004). CCKX6 (NCLX) is an essential component of the mitochondrial Na+/Ca2+ exchanger (Palty et al., 2010; Drago et al., 2011). It usually mediates mitochondrial Ca2+ extrusion (De Marchi et al. 2014). However, the mitochondrial calcium uniporter channel (MCU) and mitochondrial Na+ /Ca2+ exchanger, NCLX, mediate Ca2+ entry into and release from this organelle and couple cytosolic Ca2+ and Na+ fluctuations with cellular energetics (Verkhratsky et al. 2017).
SLC24A6 of Homo sapiens
The K+-dependent Na+/Ca2+ exchanger, MCKX4 (has 40x higher affinity for K+ than NCKX2 due to a threonine to alanine substitution at position 551 in NCKX2 (Visser et al., 2007)). NCKX4 is highly expressed and regulates Ca2+ transport in ameloblasts during amelogenesis (the formation of tooth enamel). In fact, MCKX4 is critical for enamel maturation (Wang et al. 2014). Residues involved in Na+ binding have been identified (Altimimi et al. 2010).
SLC24A4 of Homo sapiens
Trans-Golgi network K+-dependent Na+/Ca2+ antiporter SLC24A5 (NCKX5) (regulates melanogenesis; determines skin color variation) (Ginger et al., 2008).
SLC24A5 of Homo sapiens
|2.A.19.4.7||The endomembrane Ca2+:cation exchanger (CCX, CAX9 or CCX3); transports H+, Na+, K+ and Mn2+; expressed primarily in flowers (Morris et al., 2008).|
CAX9 of Arabidopsis thaliana (Q9LJI2)
K+ uptake and Na+ transporter, CCX5 (CAX11) (Zhang et al., 2011).
CCX5 of Arabidopsis thaliana (O04034)
Na+/K+/Ca2+ exchanger-1 isoform 1, NCKX-1
|Animals||SLC24A1 of Homo sapiens|
Putative Ca2+:H+ or Ca2+:Na+ antiporter with two 5 TMS internal repeats (Sääf et al. 2001).
Bacteria and Archaea
ChaB (YrbG) of E. coli
|2.A.19.5.2||Cation (Ca2+/Na+):proton antiporter, ChaA or CaxA (confers both Na+ and Ca2+ resistance) (Wei et al., 2007)||Bacteria||ChaA of Alkalimonas amylolytica (Q0ZAI3)|
Na+:Ca2+ exchanger, NCX_Mj (3-d structure known at 1.9 Å resolution; PDB# 3V5U (Liao et al., 2012). Contains 10 TMSs with two 5 TMS repeats. Four ion binding sites near the center of the protein are present, one specific for Ca2+ and three probably for Na+. Two passageways allow for Na+ and Ca2+ access from the external side. However see a more recent analysis reported for 2.A.19.8.2 (Nishizawa et al. 2013). Transport
of both Na+ and Ca2+ requires protonation of D240, but this side chain does not coordinate
either ion, implying that the ion exchange stoichiometry is 3:1 and
that translocation of Na+ across the membrane is electrogenic although transport of Ca2+ is not (Marinelli et al. 2014).
NCX_Mj of Methanococcus (Methanocaldococcus) jannaschii (Q57556)
Na+/Ca2+ exchanger. Transport is electrogenic with a likely stoichiometry of 3 or more Na+ for each Ca2+ but K+-independent (Besserer et al. 2012).
MaX1 of Methanosarcina acetivorans
Vacuolar electrogenic Mg2+, Zn2+, Fe2+, and possibly Cd2+:H+ antiporter, MHX (found in the vascular cylinder; may control the partitioning of Mg2+ and Zn2+ between plant organs). MHX porters are found only in plants and probably have 9 TMSs. Their properties have been reviewed (Gaash et al. 2013).
MHX of Arabidopsis thaliana
Low affinity vacuolar monovalent cation (Na+ (Km=20 mM) or K+(Km=80 mM)):H+ antiporter, Vnx1. (Ca2+ is not transported; plays roles in ion and pH homeostasis) (Cagnac et al., 2007)
Vnx1 of Saccharomyces cerevisiae (P42839)
Uncharacterized protein of 739 aas.
UP of Ornithorhynchus anatinus (Duckbill platypus)
Calcium:proton exchanger, CAX(CK31). The function was demonstrated by purification and reconstitution in liposomes (Ridilla et al. 2012). The protein forms dimers in the membrane but can be purified as a monomer. The dimer interface seems to involve TMSs 2 and 6 (Ridilla et al. 2012).
CAX(CD31) of Caulobacter sp. strain K31
Ca2+:H+ antiporter of 405 aas, CAX_Af. The inward facing 3-d structure has been solved to 2.3 Å resolution (Nishizawa et al. 2013). The authors compare this structure to the outward facing 1.9 Å structure of NCX_Mj (TC# 2.A.19.5.3) and suggest that Ca2+ or H+ binds to the cation-binding site mutually exclusively. The first and sixth TMSs alternately create hydrophilic cavities on the intra- and extracellluar sides of the membrane. The inward and outward-facing transitions are triggered by ion binding (Nishizawa et al. 2013).
Ca2+:H+ antiporter CAX_Af of Archaeoglobus fulgidus
Mg2+ transporter (Mg2+-specific channel-like exchanger) of 550 aas (Preston and Kung 1994; Haynes et al. 2002). Has 10 putative TMSs in a 5 + 5 TMS arrangement and exhibits properties of a channel (Haynes et al. 2002). The mutant form is called 'eccentric' and exhibits backwards swimming behavior (Preston and Kung 1994).
Ca2+-dependent Mg2+ transporter of Paramecium tetraurelia
Probable Mg2+-specific channel-like exchanger of 625 aas.
Probable Mg2+-specific channel-like exchanger of Tetrahymena thermophila