2.A.19 The Ca²⁺:Cation Antiporter (CaCA) Family

Proteins of the CaCA (NCX) 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). These nine-TMS proteins, widely distributed in the brain and in the heart, work bidirectionally. When they operate in the forward mode, they couple the extrusion of one Ca²⁺ to the influx of three Na⁺ ions, but when it operate in the reverse mode, three Na⁺ are extruded while one Ca²⁺ enters the cells. Different isoforms of NCX, named NCX1, NCX2, and NCX3, have been described in the brain, whereas only, NCX1, has been found in the heart (Annunziato et al. 2004). During the alternating-access transition, ions remain bound in the center of the protein. At the same time, two pseudo-symmetric helices slide across the lipid bilayer, opening a pathway to the binding sites from one side of the membrane while occluding access from the other (Marinelli and Faraldo-Gómez 2023). The alternating access transition requires the binding of either three Na ⁺ or one Ca ⁺ ion, as only in these occupancy states, the occluded intermediate conformations are energetically accessible. Thus, at the molecular level, the emergence of selective and active transport in this class of transporters is in agreement with the well-established 3Na⁺:1Ca⁺ stoichiometry (Marinelli and Faraldo-Gómez 2023).

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). NCXs are involved in neurodegeneration and remyelination failure in demyelinating diseases (Boscia et al. 2020).

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).

Transmembrane Ca²⁺ influx is essential to the proper functioning of the central clock in the suprachiasmatic nucleus (SCN). In SCN neurons, the clearance of somatic Ca²⁺ following depolarization-induced Ca²⁺ transients involves Ca²⁺ extrusion via Na⁺/Ca²⁺ exchanger (NCX) and mitochondrial Ca²⁺ buffering. Cheng et al. 2019 showed an important role of intracellular Na⁺ in the regulation of [Ca²⁺]_i in these neurons. The effect of Na⁺ loading on [Ca²⁺]_i was determinedwith the Na⁺ ionophore monensin and the cardiac glycoside ouabain to block Na⁺/K⁺-ATPase (NKA).The results showed that in spite of opposite effects on spontaneous firing and basal [Ca²⁺], both monensin and ouabain induced Na⁺ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K⁺-evoked Ca²⁺ transients (Cheng et al. 2019).

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).

During the alternating-access transition, ions remain bound in the center of the protein (Marinelli and Faraldo-Gómez 2023). At the same time, two pseudo-symmetric helices slide across the lipid bilayer, opening a pathway to the binding sites from one side of the membrane while occluding access from the other. Additional simulations demonstrate that the alternating access transition requires the binding of either three Na⁺ or one Ca⁺ ion, as only in these occupancy states the occluded intermediate conformations are energetically accessible (Marinelli and Faraldo-Gómez 2023).

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)