Insights into the early evolution of animal calcium signaling machinery: A unicellular point of view

Abstract

The basic principles of Ca²⁺ regulation emerged early in prokaryotes. Ca²⁺ signaling acquired more extensive and varied functions when life evolved into multicellular eukaryotes with intracellular organelles. Animals, fungi and plants display differences in the mechanisms that control cytosolic Ca²⁺ concentrations. The aim of this review is to examine recent findings from comparative genomics of Ca²⁺ signaling molecules in close unicellular relatives of animals and in common unicellular ancestors of animals and fungi. Also discussed are the evolution and origins of the sperm-specific CatSper channel complex, cation/Ca²⁺ exchangers and four-domain voltage-gated Ca²⁺ channels. Newly identified evolutionary evidence suggests that the distinct Ca²⁺ signaling machineries in animals, plants and fungi likely originated from an ancient Ca²⁺ signaling machinery prior to early eukaryotic radiation.

1. Introduction

Binding of the divalent metal calcium ion (Ca²⁺) to alter charge and hence protein conformation is one of the most extensively employed signal transduction mechanisms in life [1]. Ca²⁺ modulates nearly every aspect of cellular function in bacteria [2], protists [3], plants [4], fungi [5] and animals [1,6]. Owing to the ∼10,000-20,000-fold Ca²⁺ concentration gradient across the plasma membrane, an intricate Ca²⁺ signaling machinery is required to maintain an extremely low cytosolic Ca²⁺ concentration at ∼100 nM in resting conditions, and to raise cytosolic Ca²⁺ concentration to several hundred nM or higher upon the activation of ion channels and transporters [1,7]. The evolution of primitive Ca²⁺ binding molecules that shaped the basic principles of Ca²⁺ signaling occurred very early in the life processes of prokaryotic organisms [7-9]. In eukaryotes, intracellular organelles such as mitochondria, lysosomes and the endoplasmic reticulum emerged as intracellular Ca²⁺ compartments to likely allow better spatial and temporal regulation of Ca²⁺ concentration in response to more complex environmental stimuli [1,7-10].

In multicellular organisms, the need for intercellular communication is believed to have driven the development of versatile Ca²⁺ signaling systems to facilitate the communication and coordination between cells through chemical and electrical signals [7,8]. Different cell types possess distinct sets of Ca²⁺ signaling molecules to carry out specific physiological functions. Neurotransmitters activate ionotropic receptors at the postsynaptic membrane, inducing Ca²⁺ influx critical in the nervous system, heart, skeletal muscle, and secretory organs. Immune cells sense invading organisms and adapt via Ca²⁺-mediated changes in gene expression. The cation channels of sperm (CatSpers), a class of sperm-specific Ca²⁺ channels at the sperm flagella, trigger hyperactivated motility [11,12]. Selective expression of molecules from a large repertoire of the animal Ca²⁺ signaling machinery has evolved to accommodate animal development and physiology at the cell and organ system levels.

The transition to multicellularity observed in animals, plants and fungi likely originated independently from multiple distinct ancestral unicellular lineages [13,14]. Animals [1,6], plants [3,4,15,16] and fungi [5] exhibit marked differences in terms of the components of their Ca²⁺ signaling machineries. Animal multicellularity is generally more complex, reflected in the diversity of different cell types and intercellular communications [17,18]. Similarly, animals appear to possess a complex Ca²⁺ signaling machinery while plants and fungi seem to have adopted more simplified Ca²⁺ signaling cascades [4,5,15]. Ca²⁺ signaling might have developed through independent evolution or from the same ancient Ca²⁺ signaling network in the early evolution of eukaryotes followed by subsequent lineage-specific expansion, innovation and losses.

Recent comparative genomics of animals, fungi and plants and their unicellular sister groups has illuminated the origin and early evolution of the eukaryotic Ca²⁺ signaling machinery [3,5,15,19-25]. In this review, we address the dynamic evolutionary pattern and origin of ancestral Ca²⁺ signaling molecules in close unicellular relatives of animals and in common unicellular ancestors of Opisthokonta, a clade that contains animals, fungi and their unicellular relatives (Fig. 1). For detailed evolutionary review of Ca²⁺ signaling in plants and other photosynthetic eukaryotes [3,22], see review by Edel and Kudla in the same issue [26].

Fig. 1. Schematic representation of the eukaryotic tree of life illustrating the evolutionary relationships of select species in Unikonta and Bikonta.

The tree is inferred from the Tree of Life project (http://www.tolweb.org/) and recent studies of the eukaryotic tree [29,46]. Highlighted are the close unicellular relatives of animals, M. brevicollis and C. owczarzaki, the putative unicellular progenitor of Opisthokonta T. trahens, the marine thraustochytrid protist A. limacinum, and the basally diverging alga C. paradoxa [82].

2. The eukaryotic tree of life and the origins of multicellularity

The evolutionary root of eukaryotes appears to lie between Unikonta, the eukaryotic supergroup composed of Amoebozoa and Opisthokonta (animals and fungi), and Bikonta, the eukaryotic supergroup containing plants and algae (Fig. 1) [27-29]. Poriferans (sponges), placozoans, and cnidarians (anemones) are the morphologically simplest animal phyla, with sponges being the earliest branching lineage of animals [30]. Indeed, sponges lack a nervous system and gut, both of which are present in eumetazoans - cnidarians and bilaterians (flies, worms, sea squirts, and humans). Nevertheless, comparative analysis of the Amphimedon queenslandica genome with eumetazoan genomes revealed that sponges already developed a wide array of signaling molecules and transcription factors critical for eumetazoan development and physiology, including cell cycle regulation, cell specification, cell adhesion and immunity [30]. The Amphimedon genome also encodes homologs of the core components of post-synaptic proteins [31] and neural regulatory proteins important for neural development and nerve cell function in eumetazoans, predating the development of the nervous system in animals [30].

2.1. Close unicellular relatives of animals – choanoflagellates and filastereans

Multicellular animals likely emerged from single-celled ancestors more than six hundred million years ago [14,32]. Choanoflagellates, a group of single-celled and colony-forming eukaryotes, display striking structural and functional similarities to flagellated collar cells (choanocytes) in sponges [33]. Choanoflagellates have a spherical or ovoid cell body with a single apical flagellum surrounded by a collar of actin-filled microvilli (Fig. 2) [34]. Phylogenomics studies have shown that choanoflagellates are the closest unicellular relatives of animals; sponges and other metazoans share a common unicellular ancestor with choanoflagellates (Fig. 1) [35-37]. The filasterean Capsaspora owczarzaki is a unicellular amoeboid symbiont of the pulmonate snail Biomphalaria glabrata. C. owczarzaki is among the closest known unicellular relatives of animals besides choanoflagellates [38]. Ancestral homologs of various signaling molecules previously thought to be restricted to animals, such as receptor tyrosine kinases and transcription factors crucial for animal multicellularity and development, are present in the genomes of two choanoflagellates Monosiga brevicollis and Salpingoeca rosetta and the filasterean C. owczarzaki [33,38-40].

Fig. 2. Components of the Ca²⁺ signaling machineries in choanoflagellates M. brevicollis and S. rosetta.

The evolutionary relationship of choanoflagellates is inferred from the Tree of Life project (http://www.tolweb.org/) and recent references [36,37]. The schematic diagram depicts a choanoflagellate cell with a spherical cell body, a collar-like ring of microvilli, and an apical flagellum [34]. Compared with M. brevicollis, S. rosetta contains two more Ca²⁺ signaling molecules – ryanodine receptors and HCN channels. Abbreviations: CNG, cyclic nucleotide-gated channel; CysLoop receptors, cysteine-loop ligand-gated receptor; HCN channel, hyperpolarization-activated cyclic nucleotide-gated channel; IP₃ receptor, inositol 1,4,5-trisphosphate receptor; Letm1, leucine zipper-EF-hand containing transmembrane protein 1; MCU, mitochondrial Ca²⁺ uniporter; MICU, mitochondrial EF hand Ca²⁺ uniporter regulator; NC(K)X, Na⁺/Ca²⁺ (K⁺-dependent) exchanger; NCLX/CCX; Na⁺/Li⁺/Ca²⁺ exchanger or cation/Ca²⁺ exchanger; P2X receptor, P2X purinergic receptor channel; PMCA, plasma membrane Ca²⁺ ATPase; SERCA, sarco/endoplasmic reticulum Ca²⁺ ATPase; SPCA, secretory pathway Ca²⁺ ATPase; STIM, stromal interaction molecule; TRP, transient receptor potential channel.

In response to environmental cues, S. rosetta and C. owczarzaki can differentiate into different cell types and form multicelled colonies (or aggregative multicellularity) as part of their life cycle, which may resemble a transition state in the early evolution of animal multicellularity [41,42].

2.2. A putative unicellular progenitor of Opisthokonta - the apusozoan Thecamonas trahens

The complex multicellularity in the lineages leading to animals and fungi appears to have evolved independently from lineage-specific unicellular relatives. Apusozoa (apusomonads), a group of biflagellate and gliding protists, is believed to branch as a sister group to Opisthokonta (Fig. 1) [29,43]. The phylogenetic position of apusomonads between Amoebozoa and common ancestors of Opisthokonta makes them ideal candidates for comparative studies into the origin and early evolution of animals and fungi. The apusozoan protist Thecamonas trahens (previously known as Amastigomonas sp.) was chosen for genome sequencing as a representative outgroup to the entire opisthokont clade [44].

T. trahens was recently shown to harbour the core components of the integrin-mediated cell adhesion complex, a signaling machine critical for intercellular communication in animals [45]. Moreover, several components of the integrin adhesion complex have been lost independently in fungi and choanoflagellates. The absence of many of the integrin components in M. brevicollis and S. rosetta indicates the importance of a broad taxonomic sampling in comparative genomics of cellular signaling pathways [45].

2.3. Bikonta - the eukaryotic supergroup containing plants and algae

Comparative genomics of Ca²⁺ signaling molecules in bikont species, the eukaryotic supergroup including plants, algae and diatoms [27-29,46], has been reported previously [3,15,20,22,47,48] and is reviewed in this issue [26]. A brief discussion on the conservation of key Ca²⁺ signaling molecules will be presented based on a recent report of genomic analysis of a marine bikont protist Aurantiochytrium limacinum and a basally diverging alga Cyanophora paradoxa [25].

3. Evolution of the Ca²⁺ signaling machinery in close unicellular ancestors of animals

The core components of the Ca²⁺ signaling machinery in animals comprise proteins that control Ca²⁺ influx and extrusion across plasma or organellar membranes [1,6]. In resting cells, low cytosolic Ca²⁺ levels are maintained by plasma membrane Ca²⁺ ATPases (PMCAs) and sarco/endoplasmic reticulum Ca²⁺ ATPases (SERCAs), Na⁺/Ca²⁺ exchangers (NCXs), and cytosolic Ca²⁺ binding proteins and buffers. In response to appropriate environmental stimuli, cytosolic Ca²⁺ signals mainly arise from 1) Ca²⁺ entry across the plasma membrane through Ca²⁺ channels and/or the “reverse mode” of NCXs; 2) Ca²⁺ release from intracellular Ca²⁺ stores such as the endo/sarcoplasmic reticulum (ER/SR), through the intracellular Ca²⁺ release channels inositol 1,4,5-trisphosphate receptors (IP₃Rs) and/or ryanodine receptors (RyRs); or 3) the combination of both Ca²⁺ entry and Ca²⁺ release, such as excitation-contraction coupling in cardiac muscle and store-operated Ca²⁺ entry (SOCE) in many nonexcitable cells. In addition, endolysosomes, mitochondria and other intracellular membrane-bound compartments are important regulators of Ca²⁺ homeostasis [1,6,10].

Multicellular complexity is reflected by the presence of numerous cell types with distinct physiological functions, for example, approximately ∼200 somatic cell types in hominids [49]. Many components of the animal Ca²⁺ signaling machinery show tissue- and/or cell-type-specific expression. Several lines of evidence indicate that homologs of a wide range of signaling molecules required for animal multicellularity and development are present in unicellular ancestors of animals [13,14]. These findings prompted us to determine the evolutionary pattern and origin of the animal Ca²⁺ signaling machinery in the genomes of choanoflagellates M. brevicollis and S. rosetta (Fig. 2) and the filasterean C. owczarzaki [21,23].

3.1. Ca²⁺ entry across the plasma membrane

The choanoflagellates M. brevicollis [21] and S. rosetta [23] have all 5 modes of regulated Ca²⁺ entry across the plasma membrane identified in animals [50] — the Ca²⁺ release-activated Ca²⁺ channel (Orai) and the ER Ca²⁺ sensor protein stromal interaction molecule (STIM), ligand-gated channels (nicotinic acetylcholine receptor and P2X purinergic receptor), voltage-gated Ca²⁺ channels (CaV; similar to dihydropyridine-sensitive L-type Ca²⁺ channels), second messenger-gated channels (cyclic nucleotide-gated), and transient receptor potential (TRP) channels (Fig. 2). The Ca²⁺-permeable mechanosensitive Piezo channels are also present in M. brevicollis and S. Rosetta [25]. Furthermore, M. brevicollis contains NCXs [51].

Most TRP channels are Ca²⁺ permeable, but the degree of Ca²⁺ selectivity varies widely [52,53]. TRP channels appear to be polymodal cell sensors responding to environmental signals such as chemical compounds, changes in temperature, and pH. Homologs of 5 mammalian TRP channel families - TRPC, TRPV, TRPM, TRPML and TRPA, but not TRPP, are identified in M. brevicollis and S. rosetta [21,23]. Although TRP-like channel sequences are identified in several bikont protists [3,22,25], TRP channel homologs in choanoflagellates show higher degree of sequence conservation and are phylogenetically grouped with known animal TRP subfamilies [21,23]. It is not clear if TRPML and TRPP are on intracellular or specialized membranes as they are in higher organisms.

SOCE is a major Ca²⁺ influx pathway in nonexcitable cells, but is also present in excitable cells such as skeletal muscle [50]. ER Ca²⁺ depletion is sensed by ER-membrane spanning STIM proteins, which then bind and gate highly Ca²⁺-selective Orai channels on the plasma membrane. Orai and STIM homologs in M. brevicollis and S. rosetta possess highly conserved motifs, intragenic repeat patterns and critical residues identified in their animal counterparts [54,55]. Orai and STIM homologs are also present in C. owczarzaki [23]. SOCE may represent a primordial Ca²⁺ entry pathway in unicellular organisms. Distantly related Orai-like sequences can be occasionally found in few bikont protists [56], but these bikont protists generally lack STIM homologs and often IP₃Rs, two integral parts of animal SOCE. Therefore, Orai and STIM-mediated Ca²⁺ entry in response to ER Ca²⁺ depletion likely evolved in the ancestral animal lineages as early as the amoeboid holozoan C. owczarzaki, after the animal–fungi split. SOCE is also involved in exocytosis in Paramecium following Ca²⁺ release from alveolar sacs mediated by polyamine, caffeine or 4-chloro-meta-cresol [57-59]. However, Orai/STIM homologs are absent in Paramecium tetraurelia [56]. The molecular components of SOCE in Paramecium remain to be identified.

Ca_V channel homologs are found in choanoflagellates M. brevicollis [21] and S. rosetta [23]. In addition, cyclic nucleotide-gated channel homologs are present in M. brevicollis. A homolog of hyperpolarization-activated, cyclic nucleotide-regulated channel, which has been shown to be Ca²⁺ permeant [60], is found in S. rosetta [61]. In contrast, none of these channels are present in C. owczarzaki [23]. Moreover, P2X receptor channels are found in all vertebrates, many invertebrates, basal fungi, and certain protists. Modern fungi, land plants and some invertebrate species generally lack P2X receptor channel homologs [62,63], but P2X receptors are present in the three unicellular ancestors of animals discussed here [21,23].

3.2. Ca²⁺ channels at the ER/SR and other organellar membranes

Ca²⁺ release from the ER/SR Ca²⁺ store through IP₃Rs and/or RyRs is a common feature of almost all animal cell types [24]. IP₃Rs are ubiquitously distributed, while RyRs are enriched in skeletal and cardiac muscles and neurons [64]. The first protozoan IP₃R was characterized from the ciliate protist P. tetraurelia [65], which possess 34 IP₃R/RyR-like homologs [59,66,67]. Multiple copies of IP₃R homologs are found in M. brevicollis, S. rosetta and C. owczarzaki [21,23]. Compared with P. tetraurelia IP₃Rs, M. brevicollis IP₃Rs show overall higher sequence identity and similarity to animal IP₃Rs [21].

RyR homologs with SPRY structural domains and moderate sequence identity/similarity with animal RyRs are present in C. owczarzaki and S. rosetta [23], but not in M. brevicollis [21]. Prototype RyRs, bearing the critical protein domains such as the SPRY domain and key residues conserved in animal RyRs, likely emerged in the unicellular lineages leading to animals [23,68]. Ancestral IP₃R/RyR-like homologs are found in P. tetraurelia and several other protists [48,59,66-68]. Late incorporation of SPRY and RyR domains into ancestral homologs may have led to the innovation of animal RyRs [68].

Among the three unicellular ancestors of animals discussed here, only S. rosetta contains both Ca_V channel and RyR homologs, which are known to be functionally coupled in excitable cells of animals [64]. Four-domain voltage-gated Na⁺ (Na_V) channel homologs have also been found in choanoflagellates [69,70]. Whether an ancestral form of functional coupling between Ca_V channel and RyR homologs by membrane depolarization had evolved in S. rosetta is unknown.

Some TRP channels like TRPML are primarily localized in intracellular compartments such as lysosomes [53,71]. These intracellular TRP channels might contribute to organellar Ca²⁺ release. TRPML channel homologs are found in M. brevicollis, S. rosetta and C. owczarzaki [23].

Two-pore channels (named for having two pore domains, not for having two pores; TPCs) localise to acidic organelles. They are found in land plants, animals and many protists, but are absent in fungi and many algae [3,72]. TPCs are present in the three unicellular ancestors of animals presented here [21,23].

In mitochondria, cytosolic Ca²⁺ readily diffuses through large pores on the outer membrane, whereas Ca²⁺ flux across the inner membrane is tightly regulated by ion channels and transporters [1]. The mitochondrial Ca²⁺ uniporter (MCU and MICU subunits) proteins are widely distributed in animals, plants, basal fungi and many protists, but are absent in certain protozoan and fungal lineages [23,73]. Homologs of MCU and MICU as well as the mitochondrial Ca²⁺/H⁺ exchanger (Letm 1) and the mitochondrial Na⁺/Ca²⁺ exchanger (NCLX) are all found in M. brevicollis, S. rosetta and C. owczarzaki [23].

3.3. Removal of cytosolic Ca²⁺ signals

To prevent Ca²⁺ overload and maintain Ca²⁺ homeostasis, Ca²⁺ signals must be removed from the cytosol, either to the extracellular environment or to intracellular Ca²⁺ stores. All three unicellular ancestors of animals discussed here contain PMCA and SERCA pumps and Na⁺/Ca²⁺ exchangers that can function to maintain low cytosolic Ca²⁺ [21,23]. Both K⁺-independent Na⁺/Ca²⁺ exchangers (NCXs) and K⁺-dependent Na⁺/Ca²⁺ exchanger (NCKX) are present in M. brevicollis, whereas S. rosetta contains only NCXs and C. owczarzaki has one NCKX.

4. Evolution of the Ca²⁺ signaling machinery in the apusozoan T. trahens

Animals and fungi diverged from a common unicellular ancestor of Opisthokonta approximately one billion years ago [44]. Therefore, analyzing the genome of the apusozoan protist T. trahens, a putative unicellular progenitor of Opisthokonta, would likely reveal the common components of the distinct Ca²⁺ signaling machineries in animals and fungi. We recently identified a complex ancestral Ca²⁺ signaling network in T. trahens [23]. Not surprisingly, T. trahens contain not only many components of the animal Ca²⁺ signaling machinery, but also fungal proteins that are absent in the three unicellular ancestors of animals, including a Ca²⁺/H⁺ exchanger (CAX) and putative stretch-activated Ca²⁺ channel Mid1 homologs. CatSper channels are also present in T. trahens. Interestingly, our analysis revealed that the basal chytridiomycete fungi Allomyces macrogynus and Spizellomyces punctatus had retained P2X receptors [62], which had long been thought to have been lost in the fungal lineage [63].

Dictyostelium discoideum, a soil-living amoeba, lacks many components of the Ca²⁺ signaling system indentified in T. trahens; D. discoideum does not have homologs of SERCA and SPCA pumps, Na⁺/Ca²⁺ exchangers, Ca_V channels and CNG channels [56] or CatSpers. Amoebozoa and Apusozoan species are placed at the base of Unikonta (Fig. 1) [29]. Thus, to further explore the evolutionary origins of the animal Ca²⁺ signaling machinery requires extensive comparative genomics analyses of species in Bikonta. Previous genomics studies of plant, algal and other bikonts have provided detailed comparison of many Ca²⁺ signaling molecules between various bikont species and animals [3,15,20,22,47]. In the following section, we will review our recent findings on the early evolution of the CatSper channel complex and three classes of cation/Ca²⁺ exchangers [25], and briefly discuss the evolution of four-domain Ca_V channels.

5. Evolution of the CatSper complex, cation/Ca²⁺ exchangers, and four-domain Ca_V channels

5.1. The CatSper complex

Flagella and cilia are believed to have arisen from the last common ancestor of all eukaryotes [74]. Flagella and motile cilia are structurally similar organelles in eukaryotes, projecting from the cell surface with a typical “9+2” axoneme. Primary cilia lack the central-pair microtubules and dyneins, and thus, are immotile [74]. In humans, primary cilia are present in almost every cell type, whereas flagella are only found in sperm cells, and motile cilia are selectively expressed in few epithelial cell types. Recent electrophysiological and molecular studies revealed that distinct Ca²⁺ signaling molecules function in these compartments – CatSpers in sperm flagella (Fig. 3A) [75] and TRPP channels in primary cilia [76,77].

Fig. 3. Evolution of the CatSper channel complex and cation/Ca²⁺ exchangers.

(A) Schematic representation showing the head, midpiece and principal piece regions of mammalian spermatozoa. Located at the principle piece, the CatSper channel complex mediates Ca²⁺ influx that is critical for sperm hyperactivation. The mammalian CatSper complex is composed of four pore-forming α subunits and at least three auxiliary subunits – CatSper-β, CatSper-γ, and CatSper-δ [78]. (B) Topological illustration of cation/Ca²⁺ exchangers. NCX, NCKX, CAX, and YRBG exchangers all contain two transmembrane domains, each with 5-TM segments and one alpha-repeat. An extra N-terminal TM segment (S0) often serves as the signal peptide or targeting sequence of NCX, NCKX and CAX in eukaryotes. The key acidic residues crucial for ion exchange activity, one each in the two alpha repeats, are labelled for each exchanger class. (C) Distribution of NaChBac-like proteins, CatSpers, and cation/Ca²⁺ exchangers in select unikont and bikont species. Due to incomplete genome assembly, the exact numbers of homologs in C. paradoxa, shown as filled circles, remain to be determined. Abbreviations: CAX, cation/H⁺ exchanger; NaChBac, Na⁺ channel from Bacillus halodurans; NCKX, K⁺-dependent Na⁺/Ca²⁺ exchanger; NCX, K⁺-independent Na⁺/Ca²⁺ exchanger; YRBG, an Na⁺/Ca²⁺ exchanger in Escherichia coli.

The mammalian CatSper complex comprises four pore-forming α subunits and at least three auxiliary subunits – CatSper-β, CatSper-γ, and CatSper-δ, all of which are sperm-specific transmembrane proteins (Fig. 3A) [78]. The identification of the CatSper channel complex in T. trahens suggests that the protein complex critical for mammalian sperm hyperactivation might have an ancient role in regulating flagellar motility in protists, predating the divergence of animals and fungi. CatSper homologs were not found in previously sequenced fungal genomes [79]. The basal fungus A. macrogynus produces motile gametes with flagella. Similar to progesterone-activated Ca²⁺ entry in human sperm cells, a pheromone released by female gametes can induce Ca²⁺ influx in A. macrogynus sperm cells and modulate sperm motility [80]. Indeed, the CatSper Ca²⁺ channel complex is present in A. Macrogynus [23]. CatSpers have not been found in bikont protists such as Chlamydomonas and Paramecium, algae, and plants. However, many lower plant and algal species also have motile gametes [3], which prompted the search for CatSper channel homologs in bikont species.

Recent extensive searches of bikont genomes revealed the presence of CatSpers in Aurantiochytrium limacinum [25], a common marine thraustochytrid protist within one of the earliest diverging lineages in the stramenopile phylum [81]. Both A. limacinum and T. trahens possess the same components of the CatSper channel complex – four pore-forming α subunits, auxiliary β and γ subunits and a distantly related homolog of the δ subunit [25]. Further analysis of the preliminary Cyanophora paradoxa algal genome also identified the presence of the CatSper complex. C. paradoxa is considered to be a basally diverging alga in the lineage leading to green plants and red algae [82]. The conservation of CatSpers in at least two bikont species suggests that the CatSper channel complex likely belonged to an ancestral Ca²⁺ signaling network before the divergence of Unikonta and Bikonta. Alternatively, although much less likely, the presence of the CatSper complex in the two bikonts might be due to horizontal gene transfer from unikont species. Loss of either one of the four α subunits [83] or an auxiliary subunit [84] by gene knockout results in male infertility and degradation of the whole CatSper protein complex in mice. Assuming a similar degradation pathway in these bikonts, horizontal gene transfer would require at least seven genes encoding the CatSper complex, possibly located on different genomic regions, to be simultaneously transferred to host species. Interestingly, similar to CatSpers, TRPP channel homologs are found in both T. trahens and A. limacinum [25]. Thus, the regulatory role of CatSpers in flagellar motility and TRPP in primary cilia might have evolved at the root of early eukaryotic evolution.

Ca²⁺ signaling is critical to initiate complex flagellar activities such as capacitation, chemotaxis, and hyperactivation [78]. However, regular beating patterns of flagella depend on ATP hydrolysis by axonemal dyneins; neither CatSpers nor Ca²⁺ is required. The loss of the entire CatSper complex in several metazoan lineages likely reflects a species-dependent manner in modulating flagellar motility [79]. Hyperactivation might not be required for sperm function, which eventually led to the relaxation of selective pressure on CatSper-induced Ca²⁺ influx in sperm cells of these lineages. Similar cases of evolutionary degeneration have been documented in the loss of two other ion channels in primate evolution – TRPC2 [85] and TPC3 [86].

5.2. Cation/Ca²⁺ exchangers

A defining feature of cation/Ca²⁺ exchangers is the presence of two highly conserved α-repeat regions in the two transmembrane domains, separated by a relatively large intracellular loop [51]. NCX, NCKX and CAX are three major eukaryotic branches of the cation/Ca²⁺ exchanger superfamily [19]. Biochemical [87,88] and crystallographic [89,90] evidence indicates that these exchangers share the same overall structure with 10-TM segments, two inverted α-repeats and two key acidic residues essential for ion binding and transport (Fig. 3B). NC(K)Xs and CAXs were previously shown to be present in animals and in bacteria, plants, and fungi, respectively [19,91].

The apusozoan T. trahens contains homologs of both NCKX and CAX exchangers [23]. CAX is absent in the three unicellular relatives of animals described above and NC(K)X is not found in the two basal fungi [23], suggesting that the loss of CAX in the animal lineage and NC(K)X in the fungal lineage occurred soon after the animal/fungal divergence. A. limacinum possesses all three classes – NCKXs, NCXs and CAXs (Fig. 3C). Consistent with a recent report on the evolution of cation/Ca²⁺ exchangers in plants and algae [92], the presence of NCX, NCKX and CAX exchangers in A. limacinum suggests the common origin of these three classes of exchangers in ancestral protists prior to the Unikonta/Bikonta split. In the lineage leading to green plants, NCX and NCKX were often lost and CAX exchangers were retained as early as in the basally diverging freshwater alga C. paradoxa [25]. The presence of NCKX in many marine protists, but not in freshwater protists, may correlate with the adaptation of NCKX to regulate cytosolic Ca²⁺ in the high Na⁺, Ca²⁺, and K⁺ environment of seawater [92].

5.3. Relationship of CatSpers and cation/Ca²⁺ exchangers to their prokaryotic counterparts

The NaChBac Na⁺ channel [93] and the YRBG exchanger [51] are speculated to be the prokaryotic counterparts of the CatSper pore-forming α subunits and the cation/Ca²⁺ exchangers, respectively. Gene duplication events may have led to the expansion of four unique CatSper channel pore-forming α subunits and three classes of exchangers – NCKXs, NCXs and CAXs. YRBG-like exchangers in prokaryotes can mediate Na⁺/Ca²⁺ exchange activity with kinetic characteristics similar to those of NCXs [89,94]. NaChBac-type channels may be involved in regulating flagellar motility and chemotaxis in certain bacteria, although more basic functions in processes such as metabolism are likely [95]. While YRBG-like exchangers have yet to be found in eukaryotes [92], NaChBac-type channels with divergent P-loop sequences and presumably higher Ca²⁺ selectivity are present in two diatom genomes (Fig. 3C) [3]. Indeed, NaChBac-type bacterial channels appear to be phylogenetically closer to CatSper channels [96].

5.4. Evolution of four-domain voltage-gated Ca²⁺ channels

Four-domain Ca_V channels are an integral part of the animal Ca²⁺ signaling machinery. Similar to four-domain Na_V channels in animals, the ion-conducting pores of four-domain Ca_V channels reside in the centre of the large polypeptide that spans the plasma membrane [97]. Lined by the transmembrane (TM) segments (S) S5 and S6 and the intervening ‘Pore (P)-loop’, each domain contributes to the central pore, with the four voltage-sensing modules (S1–S4 TM helices) symmetrically arranged around the central pore. The four-domain Ca_V channels probably arose by two sequential gene duplication events from a single six-TM voltage-gated channel at the root of eukaryotes [98]. The four-domain Na_V channels likely evolved from ancestral Ca_V or Na_V channels in protists since Na_V homologs have already been identified in T. trahens [69] and M. brevicollis [70]. Similarly, Na⁺-selective NaChBac-type channels are speculated to have evolved independently from ancestral non-selective, or Ca²⁺-selective channels in bacteria [96]. It appears that all voltage-gated K⁺ (K_V), Ca_V, and Na_V channels likely arose from a single voltage sensor domain coupled to an S5/S6 pore domain. K_V channels are widely distributed in all organisms from bacteria to humans. Presumably early in eukaryotic evolution, a six-TM single-domain ancestral channel gave rise to ancient two-domain ion channels by duplication (TPCs), which then further duplicated to form evolutionary precursors of modern four-domain ion channels [96,98]. Single-domain channels such as K_V, CatSper, transient receptor potential (TRP), and NaChBac-type channels have been phylogenetically placed outside the individual domain clades of four-domain Ca_V and Na_V channels [96], raising the possibility that ancestral forms of four-domain ion channels in early eukaryotic evolution might have been lost or functionally diversified to mask their ancestral state.

The crystal structures of NaChBac-type bacterial Na_V channels revealed that the side groups of key Glu residue (E/E/E/E, one from each 6-TM subunit) coordinate with other residues in the P-loop to form the strongly negatively charged portion in the selectivity filter [99,100]. Mutagenesis studies have shown that a point mutation of Glu to Asp (D/D/D/D) yields a mutant NaChBac channel permeable to both Ca²⁺ and Na⁺, and the incorporation of two additional Asp residues in the P-loop region converts NaChBac from a highly Na⁺-selective channel to a relatively Ca²⁺-selective channel [101]. In addition, mutations in the selectivity filter of the animal four-domain Ca_V and Na_V channels also alter their ion selectivity [102,103]. Therefore, the P-loops of ancestral four-domain Ca_V and Na_V channels likely underwent evolutionary selection pressures to modulate ion selectivity in the lineages leading to fungi and animals. Recent phylogenetical and pharmacological analyses of TPCs and TPC-related proteins in unicellular organisms demonstrated that modifications in ion selectivity within four-domain Ca_V and Na_V channels may have occurred before intragenic duplication of an ancient two-domain ancestor [104].

Finally, analysis of four-domain voltage-gated ion channel sequences from Opisthokonta and T. trahens suggests that fungal Ca²⁺ channels and animal Na⁺ leak- nonselective channels, both of which are voltage-insensitive, diverged from Ca_V and Na_V channels before the animal-fungal split [105]. Furthermore, in contrast to the analysis of several putative Na_V/Ca_V-like four-domain channel sequences in T. trahens [69,105], all Na_V and Ca_V channel homologs from choanoflagellates M. brevicollis and S. rosetta form clear branching patterns with their animal counterparts [21,23]. These findings further support the lineage-specific modulation of P-loop sequence and ion selectivity in the animal and fungal lineages after divergence.

6. Conclusion

Comparative genomics of recently sequenced genomes of unicellular organisms have provided evolutionary evidence that the distinct Ca²⁺ signaling machineries in animals, plants and fungi share a common origin in ancestral protists prior to the eukaryotic radiation (Fig. 4) [3,22-25]. These mechanisms appear to be comprehensive, as shown in the very few protists such as T. trahens and A. limacinum, and are often conserved in the lineage leading to animals with further diversification and expansion. Even though most modern protists, plants and fungi tend to exhibit simplified Ca²⁺ signaling networks, traces of relatively conserved Ca²⁺ signaling systems remain in some basally divergent species, for instance, the basal chytridiomycete fungus A. macrogynus [23] and the basally diverging alga C. paradoxa [25].

Fig. 4. Schematic representation of the evolutionary history of Ca²⁺ signaling machineries in eukaryotes.

A comprehensive Ca²⁺ signaling machinery likely had evolved early at the root of eukaryotes before the divergence of major eukaryotic supergroups. In the lineage leading to animals, the ancestral Ca²⁺ signaling machinery is often highly conserved. In contrast, the Ca²⁺ signaling machineries in most modern protists, plants and fungi exhibit substantial gene losses. However, evidence of the presence of an extensive ancestral Ca²⁺ signaling machinery can still be found in few protists such as A. limacinum and T. trahens.

Questions of current interest include functional characterization of these ancestral homologs and investigation of their physiological roles in the unicellular lineages leading to animals. Further studies on the extensive network of Ca²⁺ signaling molecules in unicellular organisms will provide novel evolutionary and mechanistic insights into the formation of distinct Ca²⁺ microdomains in a single cell to mediate coordinated interaction and spatial and temporal patterns of different Ca²⁺ signals.