... likely made early gnathostomes fierce predators and the dominant vertebrates in the ocean. Later in vertebrate evolution, the Nav channel gene family expanded in parallel in tetrapods and teleosts (~9 to 10 genes in amniotes, 8 in teleosts). This expansion occurred during or after the late Devonian ...

... Section of Neurobiology, University of Texas, Austin, TX 78712; and Marine Biological Laboratory, The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Woods Hole, MA 02543. E-mail: h.zakon@...

... (electric fish), in protection against lethal Nav channel toxins (snakes, newts, pufferfish, insects), and in specialized habitats (naked mole rats)....

... Multicellular animals evolved >650 million years ago (Love et al., 2009). The nervous system and muscles evolved shortly thereafter. The phylogeny of basal metazoans is poorly resolved, likely because of the rapid radiation of these then-new life- ... al., 2005), so depending on the phylogeny one embraces, the nervous system evolved once with a loss in sponges, or twice independently in ctenophora and bilateria + cnidaria or bilateria and cnidaria + ctenophora (Moroz, 2009; Schierwater et al., 2009). However, in all animals with nervous systems, ... generate action potentials (APs), release excitatory and inhibitory neurotransmitters, form circuits, receive sensory input, innervate muscle, and direct behavior....

... The history of brain evolution and its key neural genes would fill volumes. I will use voltage-dependent Na+ (Nav, Na-permeable voltage-dependent = protein; scn, sodium channel = gene) ... as an exemplar to tell this story because all neuronal excitability depends on Nav channels, there is a good understanding of their function and regulation from biophysical, biochemical, and modeling studies, and there are fascinating examples of ecologically relevant adaptations. An ... rationale is that although many proteins, such as immunoglobins, sperm and egg receptors, olfactory receptors, opsins, and surface proteins of pathogens, are routinely studied in the field of molecular evolution, only recently have ion channels begun to receive greater ...

... Voltage-gated ion channels are the basis of electrical excitability of all animals and many single-celled eukaryotes. Potassium leak and voltage-dependent K+ (Kv) channels appeared 3 billion years ago in bacteria and occur in all organisms (Anderson and Greenberg, 2001) (Fig. 2.1). ... establish resting potentials and repolarize membranes after excitatory events. Kv channels are the “founding members” of the family of ion-permeating channels whose basic ...

... figure is the structure of the channels moving from left to right showing a linear leak K+ channel that is composed of two membrane-spanning helices and a pore (blue), a 6TM channel with a single voltage sensor (red), and four domain 4×6TM channels with four voltage sensors. There is uncertainty ... the origin of the 4×6TM family, which more likely evolved in eukaryotes than prokaryotes, as indicated in this figure. A more precise and detailed relationship among Cav and Nav channels in basal metazoans and their sister group, the choanoflagellates, is given in Fig. 2.3. Reprinted ... Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 129/1, Peter A. V. Anderson, Robert M. Greenberg, Phylogeny of ion channels: Cues to structure and function, 12-17, Copyright (2001), with permission from Elsevier. [Note: ...

... localized in endosomes and lysozomes (Galione et al., 2009). The gene for a two-domain channel likely duplicated to make a protein with four domains capable of forming a ... on its own (4×6TM). Eventually such a four-domain channel evolved (or retained) permeability to Ca2+, and these handily became involved in intracellular signaling. Other Ca2+-binding proteins and enzymes first appeared in single-celled eukaryotes (Cai, 2008). ... , there are single 6TM Na+-permeable channels localized in endosomes and lysozomes (Galione et al., 2009). The gene for a two-domain channel likely duplicated to make a protein with four domains capable of forming a ... on its own (4×6TM). Eventually such a four-domain channel evolved (or retained) permeability to Ca2+, and these handily became involved in intracellular signaling. Other Ca2+-binding proteins and enzymes first appeared in single-celled eukaryotes (Cai, 2008). ...

... bacteria (Koishi et al., 2004). Their relationship to eukaryotic Nav channels is unclear, and they will not be discussed in this review....

... The three main types of Cav channels are L, N/P/Q/R, and T. Generally speaking, L-type channels are found in muscle and neuronal dendrites, and N/P/Q/R are found in synaptic terminals and regulate transmitter release, whereas T types, which are sensitive to voltages close to resting potential, ... underlie spontaneous firing and pacemaking. These three subfamilies appear early in animals in a common ancestor of bilateria and cnidaria (Liebeskind et al., 2011) (Fig. 2.2). Choanoflagellates, single-celled protists that are the sister group to metazoans, and sponges have a ... Cav channel gene that is ancestral to the L and N/P/Q/R families. The origin of the T-type channels is not clear....

... Nav channel. Top: The Nav channel is composed of four repeating domains (I–IV), each of which has six membrane-spanning segments (S1–S6), and their connecting loops (in white). Middle: The four domains cluster around a pore. Bottom: The four P loops dip down into the membrane and line the ...

... Nav channels share the 4×6TM structure (Figs. 2.1 and 2.2) with Cav channels, and it has been suggested that Nav channels evolved from Cav channels (Hille, 2001). Analysis of putative Cav and Nav channel genes from fungi, ... , and metazoans confirm this speculation and show that choanoflagellates have a channel that groups with recognized Nav channels with strong support (Fig. 2.3). The selectivity filter of 4× ... channels depends on a single amino acid in each of the four domains that come together and face each other, presumably forming the deepest point in the pore. The selectivity filter of the choanoflagellate and other basal metazoans (DEEA) is ... between bona fide Cav (EEEE) and Nav1 (DEKA) channel pores and lives on in metazoans in a Nav channel found only in invertebrates (Nav2) (Zhou et al., 2004) (Fig. 2.3). This pore sequence and studies of the ... Nav2 suggest that the choanoflagellate Nav channel is likely permeable to both Ca2+ and Na+ and may not be a pure Na+-selective channel. This will be determined when the choanoflagellate Nav channel is expressed and studied in detail....

... sedentary anemones (anthozoa). The selectivity filter DKEA enhances Na+ selectivity less than DEKA but more than DEEA (Schlief et al., 1996; Lipkind and Fozzard, 2008). The nervous system of jellyfish has clusters of neurons approaching a real central nervous system, whereas that of anemones is more ... a nerve net. Thus, enhanced Na+ selectivity occurred in parallel in medusozoan and bilaterian Nav channels along with increasing structural complexity of the nervous system (Liebeskind, 2011)....

... nervous system for rapid communication across distant parts of organisms, but that they did so by marshalling an ion that was abundant in the ocean and would minimally perturb intracellular Ca2+ levels and, therefore, intracellular signaling (Hille, 2001)....

... Besides the obvious change from Ca2+ to Na+ permeability, other changes occurred as well. The short intracellular loop between domains III and IV evolved function as the inactivation “ball” (West et al., 1992). In voltage-dependent K+ channels all four voltage sensors must be & ... + channel, activation is accomplished by the three voltage sensors in domains I–III; the voltage sensor in domain IV initiates inactivation (Chanda and Bezanilla, 2002; Chanda et al., 2004). No Cav channel has been examined in such a way that we do not know whether they also have equivalently ...

... Maximum likelihood phylogeny of the voltage-gated sodium channel family. The common ancestor of choanoflagellates (represented by Monosiga in green) and animals had a Nav channel that was likely permeable to Ca2+ and Na+ (pore motif = DEEA). This motif is present in the Nav channels of anthozoan ... (anemones, coral) and the Nav2 channel of invertebrates. The presence of a lysine (K) in the pore improves Na+ selectivity (indicated by red star). A lysine is found in ... Nav1 channels of bilaterians (DEKA) and Nav channel of medusozoan cnidaria (jellyfish) (DKEA), both of which have more centralized nervous systems than anthozoans and are motile. ...

... EVOLUTION OF NA+ CHANNEL CLUSTERING AT THE AXON INITIAL SEGMENT AND THE NODES OF RANVIER...

... Myelination and saltatory conduction are key innovations of the vertebrate nervous system that markedly increase axonal conduction velocity [myelination evolved ... times in some invertebrate lineages as well despite a widespread and persistent belief to the contrary (Hartline and Colman, 2007; Wilson and Hartline, 2011)]. Myelination is not present in agnathans but occurs in all gnathostomes, likely appearing first in a placoderm ancestor (Zalc et al., ... the adjacent node to threshold. KCNQ-type K+ channels, which help to repolarize the AP, cluster at nodes as well, both channels tethered to ankyrin and thence to the cytoskeleton....

... Remarkably, both Nav and KCNQ K+ channels evolved the same specific nine-amino acid motif for ankyrin binding (Hill et al., 2008). This motif first appears in the Nav ... of ascidians and agnathans and, indeed, Nav channels cluster at axon initial segments (AIS) in the lamprey. In lampreys, and presumably nonvertebrate chordates, the high-density clustering of Nav channels adjacent to the soma ensures sufficient current injection into the ... axon (Boiko et al., 2003). This emphasizes the distinction between older non–myelin-dependent mechanisms for clustering Nav channels at the AIS and more recent myelin-dependent clustering of Nav channels at nodes. A surprising observation is that the AIS is mobile, moving toward the soma when a ... ’s firing rate is low and away from the soma when it is high (Grubb and Burrone, 2010). This is likely different from the nodes of Ranvier, which are smaller and constrained by the myelin sheath. However, this remains to ...

... construction of the nodes of Ranvier were in place. By this time the key genes for myelin components had also evolved (Schweigreiter et al., 2006; Li and Richardson, 2008)....

... MAKING UP FOR LOST TIME: VERTEBRATE NAV CHANNEL GENES DUPLICATED EXTENSIVELY IN TELEOSTS AND TETRAPODS...

... Invertebrates have two Nav channel genes, Nav1 and Nav2, each in single copy. We have little information on the normal physiological role of...

... Nav2 channels in invertebrates [knockouts in Drosophila are not lethal and produce only a mild phenotype (Stern et al., 1990; Kulkarni et al., 2002)]. It is interesting that both genes have been lost in nematodes (Bargmann, ... ), most of which are small and depend on passive transmission of electrical activity. The predominant Nav channel gene in invertebrates (para in Drosophila), and the only Nav ...

... insight in 1970, Susumu Ohno suggested that vertebrates underwent two rounds of whole-genome duplication (WGD) at their origin (2R hypothesis) and that a subsequent third WGD occurred in teleost fishes (3R) (Ohno, 1970). Ohno believed that these ploidy events provided the raw genetic material ... which emerged many of the defining features of vertebrates. Although originally controversial, his view has been empirically confirmed (Meyer and Schartl, 1999; Jaillon et al., 2004). Nav1 channel genes show a perfect read-out of this history. A single Nav1 channel gene is present in tunicates, ... in lampreys, four in elasmobranchs and in the common ancestor of teleosts and tetrapods (Lopreato et al., 2001; Novak et al., 2006; Widmark et al., 2011; Zakon et al., 2011). As expected from a teleost-specific WGD, eight Nav ...

... However, further gene duplication/retention occurred in tetrapods above and beyond that predicted by 2R. Two of the four Nav channel genes of our tetrapod ancestors underwent a series of tandem duplications in early amniotes, ... that the stem reptilian ancestor of modern-day reptiles, birds, and mammals had nine Nav1 channel genes (Widmark et al., 2011; Zakon et al., 2011). A final duplication occurred early in the mammalian lineage, giving ...

... duplicate genes in tetrapods adaptive? We can approach this by comparing the fates of Nav channel genes with other genes in tetrapods throughout 2R and beyond. In tetrapods, the genes surrounding the Nav channel genes that would have duplicated along with them in 2R show little or no evidence of ... duplication and retention; indeed, some show a loss of one or more 2R duplicates (Fig. 2.4). This pattern of duplication and retention of Nav channel genes is statistically significantly different compared with that of the immediately surrounding genes (Zakon et al., 2011). ... superfamily, were also more likely to be lost than retained (Widmark et al., 2011). Furthermore, an analysis of Cav, transient receptor potential, and various K+ channel subfamilies shows that there was no widespread duplication and retention of other ion channel genes in the tetrapod 6TM family ...

... FIGURE 2.4 The Nav channel gene family underwent an expansion in parallel in teleosts and tetrapods. (A) A schematic chromosome with Nav channel genes (SCN, sodium channel) surrounded by other genes. (B) This chromosome, along with all of ... chordate chromosomes, duplicated twice at the origin of vertebrates (2R). (C) There was an additional round of genome duplication in teleosts (3R) and (D) tandem duplications of Nav channel genes in ancestral tetrapods and amniotes. There is no indication of any loss of Nav channel genes despite ... of surrounding genes in both teleosts and tetrapods. Furthermore, although not shown here, no other ion channel gene family duplicated after the teleost and tetrapod divergence. Thus, there ... Jenny Widmark, Görel Sundström, Daniel Ocampo Daza, Dan Larhammar, Differential evolution of voltage-gated sodium channels in tetrapods and teleost fishes, Molecular Biology and Evolution, 2011, by permission of Oxford University Press. [Note: Figure can be viewed in color in the PDF ...

... in the electric organs of two independently derived lineages of weakly electric fish. Two paralogous genes, (A) scn4aa, which encodes Nav1.4a, and (B) scn4ab, which encodes Nav1.4ab, are expressed in the muscles of teleost fish. In the two lineages of weakly electric fishes, the mormyroidea and ... , the gene for Nav1.4a (scn4aa) lost its expression in muscle and is only expressed in the electric organ. Nav1.4a underwent a burst of accelerated evolution at the origin of each lineage of electric fish. Nav1.4b, ... is expressed in muscle and may also be expressed in the electric organ, evolved at a lower rate. The rate of nonsynonymous substitutions/nonsynonymous sites/rate of synonymous ...

... and tetrapods to preserve them. Future work detailing where Nav channels are expressed and how they behave in ray-finned fish, lungfish, and nonmammalian tetrapods will shed light on this question....

... The addition of new Nav channels to the existing repertoire likely realized two benefits: enhanced computational ability and increased energetic efficiency. For example, Nav1.1 is expressed in fast-firing inhibitory cortical interneurons, and its properties allow these ... positive than Nav1.6, is found more proximally. This will ensure that APs that are first generated in the most distal AIS propagate down the axon and these are followed by APs generated in the proximal AIS that backpropagate into the soma (Hu et al., 2009)....

... should completely inactivate before the K+ channels open to minimize use of the ATP-dependent Na+/K+ pump (Hasenstaub et al., 2010; Schmidt-Hieber and Bischofberger, 2010; Sengupta et al., 2010). It is possible that variation in the properties of Nav channels allows more precision in matching their ...

... ion channels cause behavior indirectly by triggering muscle movements. Weakly electric fish, however, emit electric signals directly into the water, and these are shaped by the biophysical properties of Nav and Kv channels in their electric organs. In nonteleost vertebrates the Nav channel Nav1.4 is ... in muscle; because of the teleost-specific WGD, teleosts have two paralogs, Nav1.4a and Nav1.4b, in their muscles (Zakon et al., 2006; Arnegard et al., 2010) (Fig. 2.5). There must...

... low rates of sequence evolution and hot colors represent high rates. The arrows indicate where Nav1.4a gene expression was lost from muscle in both lineages. The production of either a ...

... years of teleost history. The only exceptions are two lineages of weakly electric fishes. These two groups—the South American gymnotiforms and African mormyriforms—evolved electric organs independently. In both lineages the gene for Nav1.4a (scn4aa) lost its expression in muscle and ... ; in some cases the same or neighboring amino acids changed in both groups. Although these substitutions have not yet been introduced into a channel and their effects tested, the implication is that these substitutions have facilitated the diversity of species-specific signals in these fish. An ...

... other lineages of fishes. For example, rapidly contracting sound-producing muscles evolved independently in at least three lineages of fishes (Bass and Ladich, 2008), and heater muscles that no longer contract but that engage in futile Ca2+ cycling to generate heat, in two lineages (Block et al., ... ). It would be intriguing to know whether Nav channels show a similar pattern of compartmentalized expression and rapid evolutionary change in specialized muscles and muscle-derived organs of these lineages. Has the duplication of a muscle-expressing Nav channel ...

... organisms (Lee et al., 2000). In any event, unlike peptide toxins that are sequestered within a gland, TTX passes through cell membranes so that although it may be concentrated in certain tissues, all tissues are more or less exposed to it (Williams ... that sequester high concentrations of TTX have evolved mechanisms to protect themselves from its effects (Kidokoro et al., 1974; Flachsenberger and Kerr, 1985). Because invertebrates possess only a single Nav channel gene, TTX resistance could occur easily enough with a single amino acid ...

..., sequester TTX. This is a general trait of tetraodontiform fishes of which there are more than 120 species. Sequencing of Nav channel genes from Fugu and other pufferfishes shows that many of the same TTX-resistant amino acid substitutions have occurred multiple times in various Nav channels and ... the fact that certain substitutions were present in ancestral tetraodontids, with other substitutions appearing in different lineages of pufferfish and in different Nav channels (Jost et al., 2008)....

... Some of the most remarkable work in this field concerns the rich and extensively studied garter snake–newt system. Newts such as the California newt (Taricha torosa) sequester high levels of TTX for protection ... predators. However, in some regions in the Pacific Northwest and northern California, the common garter snake (Thamnopis sirtalis) overlaps with some populations of the newt. Garter snakes that do not overlap with ... newts are severely affected by ingesting newts and will vomit up the newt if they are lucky and die if they are not. However, populations of garter snakes sympatric with the newts are resistant to TTX and handily take newts. Variation in the ... resistance has evolved multiple times in populations of other species of garter snakes that are also sympatric with Taricha in the Pacific Northwest and California, as well as other snake species sympatric with other newts or frogs that use TTX in South America and Asia (Feldman et al., 2009, 2012). ... , sequencing and testing of expressed Nav channels (Nav1.4, a muscle-expressing Nav...

... channel encoded by the scn4a gene) have highlighted that these channels show amino acid substitutions in the pore where TTX binds (Geffeney et al., 2005; Feldman et al., 2012). Not surprisingly, the Nav channels of the newts ...

... However, this story is richer still. Newts lay their eggs in streams and ponds, and these eggs hatch into gill-bearing larvae. The larvae do not produce much TTX. Adults, however, do. The adults are carnivorous and may be ... . Larval newts that are “downwind” of adults will flee if they smell TTX wafting toward them in the water (Zimmer and Ferrer, 2007). Thus, TTX is used as a chemical signal [it is similarly used as an attractive pheromone in pufferfish, in which males can detect ...

... most vertebrate predators because of their high titer of TTX. Nevertheless, caddis fly (Limnephilus flavastellus) larvae have evolved TTX resistance and will eat newt eggs (Gall and Brodie, 2011). It is not yet known whether this is due to a substitution in the pore of the Nav channel. Given that ... have only a single Nav channel gene, this seems likely, and it will be interesting to see whether other invertebrate egg-predators are resistant to TTX....

... Naked mole rats (Heterocephalus glaber) live at high density in subterranean tunnels and seldom emerge into the light. They have evolved a number of adaptations for this life history, among them insensitivity to acid (Park et al., 2008). ... their tunnels make carbonic acid; humans exposed to these levels of CO2 report stinging pain. However, naked mole rats show no pain-related behaviors and their C-fiber nociceptors are not activated by acid. Molecular and physiological examination of the naked mole rat’s acid-sensing (ASIC) and ... , showed no unusual behavior in these animals. Insofar as protons are also small monovalently positively charged molecules, these interact with and block Na+ channels. The Nav channel Nav1.7 sets the threshold for firing of C-fiber nociceptors. Naked mole rat Nav1.7, indeed, is extremely ... to proton block, ensuring that, at low pH, Nav1.7 will be blocked and the C-fiber nociceptors are not activated (Smith et al., 2011)....

... One unintended consequence of the liberal and worldwide use of dichlorodiphenyltrichloroethane, pyrethrin, and pyrethroid insecticides has been the rapid, massively parallel evolution of resistance to these pesticides in insects (Taylor et al., 1993; Liu et al. ... of the insecticide, were evident in the early 1950s. These insecticides target the Nav1 channels of insects. They cross the cell membrane and lodge in a hydrophobic pocket in the inner mouth of the channel, where they are believed to prevent the inactivation gate (domain III–IV linker) ... to counteract the effects of insecticides. An example of the latter is a substitution that causes the channel to open at more positive potentials and to enhance the rate at which Nav channels enter closed-state inactivation. This minimizes the number of open channels counteracting the prolonged ...

... CONCLUSIONS AND FUTURE DIRECTIONS...

... Like many key components of the nervous system, Nav channels existed before neurons. It is likely that the Nav channels of choanoflagellates and early metazoans were permeable to both Na+ and Ca2+ and evolved enhanced selectivity to Na+ in parallel in early bilaterians and jellyfish. Although ... only a single Nav1 channel gene, it is worth scouring the wealth of new genomes to determine whether there are any lineage-specific duplications, and if so, what this might mean. Further, we have little information on the Nav2 channels of invertebrates....

... The parallel expansion of Nav channel genes in tetrapods and teleosts occurred along with an increase in the number of telencephalic nuclei in both groups. This was coincident with or just after the great ... extinction, during which teleosts began their domination of the aquatic and tetrapods of the terrestrial habitats. More types of Nav channels may allow for more sophisticated computational possibilities and energy savings....