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2
Adaptive Evolution of
Voltage-Gated Sodium Channels:
The First 800 Million Years
HAROLD H. ZAKON
Voltage-gated Na+-permeable (Nav) channels form the basis for electrical
excitability in animals. Nav channels evolved from Ca2+ channels and
were present in the common ancestor of choanoflagellates and animals,
although this channel was likely permeable to both Na+ and Ca2+. Thus,
like many other neuronal channels and receptors, Nav channels predated
neurons. Invertebrates possess two Nav channels (Nav1 and Nav2),
whereas vertebrate Nav channels are of the Nav1 family. Approximately
500 Mya in early chordates Nav channels evolved a motif that allowed
them to cluster at axon initial segments; 50 million years later with the
evolution of myelin, Nav channels “capitalized” on this property and
clustered at nodes of Ranvier. The enhancement of conduction velocity
along with the evolution of jaws 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 extinction, when teleosts and
tetrapods each diversified in their respective habitats, and coincided
with an increase in the number of telencephalic nuclei in both groups.
The expansion of Nav channels may have allowed for more sophisticated
neural computation and tailoring of Nav channel kinetics with potassium
channel kinetics to enhance energy savings. Nav channels show adaptive
sequence evolution for increasing diversity in communication signals
Section of Neurobiology, University of Texas, Austin, TX 78712; and Marine Biological Labo-
ratory, The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution,
Woods Hole, MA 02543. E-mail: h.zakon@mail.utexas.edu.
21
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22 / Harold H. Zakon
(electric fish), in protection against lethal Nav channel toxins (snakes,
newts, pufferfish, insects), and in specialized habitats (naked mole rats).
M
ulticellular 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-forms (Rokas
et 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 ner-
vous systems, neurons 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) channels as an exemplar
to tell this story because all neuronal excitability depends on Nav chan-
nels, 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 additional 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 attention (Lopreato et al., 2001;
Geffeney et al., 2005; Zakon et al., 2006, 2011; Arnegard et al., 2010; Liu et
al., 2011); of these studies, the majority are on Nav channels.
SODIUM CHANNEL GENES ARE LATECOMERS TO THE
6TM FAMILY OF VOLTAGE-DEPENDENT ION CHANNELS
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). They
establish resting potentials and repolarize membranes after excitatory
events. Kv channels are the “founding members” of the family of ion-
permeating channels whose basic structure is a protein of six transmem-
brane helices (6TM) that associate as tetramers to form a channel. At some
point early in eukaryote evolution, the gene for a 6TM channel likely
duplicated, giving rise to a protein with two domains. These proteins then
dimerized to form a complete channel (Strong et al., 1993). Such a chan-
nel still exists in the two-pore channel family of Ca2+-permeable channels
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Adaptive Evolution of Voltage-Gated Sodium Channels / 23
FIGURE 2.1 Schematic diagram of the evolutionary relationships among some
key families in the ion channel superfamily. On the top of the figure is the struc-
ture 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 4x6TM channels with
four voltage sensors. There is uncertainty about the origin of the 4x6TM 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 from 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 permis-
sion from Elsevier. [Note: Figure can be viewed in color in the PDF version of this
volume on the National Academies Press website, www.nap.edu.]
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 channel on its own (4x6TM). 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). Additionally, there are single 6TM Na+-permeable channels in
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24 / Harold H. Zakon
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. Gen-
erally speaking, L-type channels are found in muscle and neuronal den-
drites, and N/P/Q/R are found in synaptic terminals and regulate trans-
mitter 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
single 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.
FIGURE 2.2 Hypothetical secondary structure of a 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 outer mouth of the channel that is evident
in an en face view of a single domain. The black dot represents the single amino
acid at the deepest position of each of the four P loops that determines Na + ion
selectivity. From Liebeskind et al. (2011).
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Adaptive Evolution of Voltage-Gated Sodium Channels / 25
Nav channels share the 4x6TM 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, choanoflagellates, 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 4x6TM
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 midway 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 invertebrate Nav2 suggest that the choano-
flagellate 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.
The presence of a K in domain III of the pore, as in the bilaterian
Nav1, increases Na+ selectivity substantially (Fig. 2.3). There is a K in
domain II in the Nav channel pore of motile jellyfish (medusozoa) but not
in 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 of a nerve net. Thus, enhanced Na+ selectivity occurred
in parallel in medusozoan and bilaterian Nav channels along with increas-
ing structural complexity of the nervous system (Liebeskind, 2011).
There is little question as to the adaptive advantage conferred by Na+-
selective channels in early animals. It was not only that, with the advent of
multicellularity, they fulfilled the need in a newly evolved 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 “engaged”
for the channel to open. In the Na+ 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 acting voltage sensors or whether
the voltage sensor in domain IV had already evolved a novel function.
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26 / Harold H. Zakon
FIGURE 2.3 Maximum likelihood phylogeny of the voltage-gated sodium chan-
nel family. The common ancestor of choanoflagellates (represented by Monosiga
in green) and animals had a Nav channel that was likely permeable to Ca 2+ and
Na+ (pore motif = DEEA). This motif is present in the Nav channels of anthozoan
cnidaria (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 the 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. Additionally, there is strong
conservation of a hydrophobic (blue) triplet of amino acids in the “inactivation
gate” region. From Liebeskind et al. (2011). [Note: Figure can be viewed in color in
the PDF version of this volume on the National Academies Press website, www.
nap.edu.]
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Adaptive Evolution of Voltage-Gated Sodium Channels / 27
EVOLUTION OF NA+ CHANNEL CLUSTERING AT THE
AXON INITIAL SEGMENT AND THE NODES OF RANVIER
Myelination and saltatory conduction are key innovations of the ver-
tebrate nervous system that markedly increase axonal conduction veloc-
ity [myelination evolved multiple 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 pres-
ent in agnathans but occurs in all gnathostomes, likely appearing first in
a placoderm ancestor (Zalc et al., 2008). Saltatory conduction depends
on high densities of Nav channels at the nodes of Ranvier that inject suf-
ficient current into the axon to depolarize 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 channels 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 high-resistance axon in the face of current shunting by
the low-resistance soma (Kole et al., 2008). Shiverer mice, which have a
mutation that prevents the formation of compact myelin, retain a high
density of Nav channels (Nav1.6) at the AIS but not along 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 surpris-
ing observation is that the AIS is mobile, moving toward the soma when a
neuron’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 be investigated.
KCNQ channels only occur in gnathostomes. Once KCNQ channels
appeared, all of the molecular components for 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
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28 / Harold H. Zakon
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,
1998), 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 channel gene in vertebrates, is Nav1.
However, Nav1 has duplicated in vertebrates.
In a prescient insight in 1970, Susumu Ohno suggested that verte-
brates 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 from 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, two 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 channel genes
are found in teleosts (Fig. 2.4).
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 duplica-
tions in early amniotes, so 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 us 10 Nav channel genes.
Was the retention of these 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 further 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). A similar analysis in teleosts shows
that nearby genes, such as members of the TGF-β receptor 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 since
the teleost–tetrapod divergence (Zakon et al., 2011). Thus, we infer that
selection acted on the Nav channel duplicates independently in teleosts
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FIGURE 2.4 The Nav channel gene family underwent an expansion in parallel in teleosts and tetrapods. (A) A schematic chro-
mosome with Nav channel genes (SCN, sodium channel) surrounded by other genes. (B) This chromosome, along with all of the
other ancestral 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 losses 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 was
likely to be strong selection for the preservation of Nav channel gene duplicates. Reprinted from 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
version of this volume on the National Academies Press website, www.nap.edu.]
29
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30 / Harold H. Zakon
FIGURE 2.5 Nav1.4a is a fast-evolving Nav channel expressed in the electric
organs of two independently derived lineages of weakly electric fish. Two paralo-
gous 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 gymnotiformes, 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, which is expressed in muscle and may also be expressed
in the electric organ, evolved at a lower rate. The rate of nonsynonymous substi-
tutions/nonsynonymous sites/rate of synonymous substitutions/synonymous
site (dN/dS) in each gene is shown by a color scale in which cool colors represent
continued
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Adaptive Evolution of Voltage-Gated Sodium Channels / 31
and tetrapods to preserve them. Future work detailing where Nav chan-
nels 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 ener-
getic efficiency. For example, Nav1.1 is expressed in fast-firing inhibitory
cortical interneurons, and its properties allow these neurons to fire at
sustained high rates (Ogiwara et al., 2007). In pyramidal neurons, Nav1.6
is found in the distal part of the AIS, whereas Nav1.2, which activates at
voltages around 20 mV more positive than Nav1.6, is found more proxi-
mally. 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).
The extent to which Na+ channels inactivate before K+ currents com-
mence influences energy consumption; optimally Nav channels 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
inactivation with Kv channels to save energy.
ADAPTIVE EVOLUTION OF NAV CHANNELS:
WEAKLY ELECTRIC FISH
In most organisms ion channels cause behavior indirectly by trigger-
ing 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 expressed 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 highly regular wave type or an irregular pulse type of
electric organ discharge is indicated in both groups. In both lineages of electric
fishes, the electric organ develops from muscle (myogenic), except for one group
(Apteronotidae) in which it is derived from the axons of motorneurons. From
Arnegard et al. (2010). [Note: Figure can be viewed in color in the PDF version of
this volume on the National Academies Press website, www.nap.edu.]
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32 / Harold H. Zakon
be strong selection for the retention of the expression of both paralogs in
muscle because they are both expressed in muscles of most teleosts exam-
ined. In other words, the expression of both genes in fish muscle remains
after 250 million years of teleost history. The only exceptions are two lin-
eages of weakly electric fishes. These two groups—the South American
gymnotiforms and African mormyriforms—evolved electric organs inde-
pendently. In both lineages the gene for Nav1.4a (scn4aa) lost its expression
in muscle and became compartmentalized in the electric organ. Nav1.4
in mammals is under strong purifying selection because mutations in the
gene for this channel often cause muscle paralysis or myotonia. Freed from
its constraints, Nav1.4a underwent a burst of evolutionary change at the
origin of both groups of electric fishes, with numerous substitutions in
key regions of the channel, many involved in inactivation (Zakon et al.,
2006; Arnegard et al., 2010). The pace of evolutionary change quickened
in similar regions of the channel in both groups; in some cases the same or
neighboring amino acids changed in both groups. Although these substi-
tutions 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 unanswered question is this: if
nonelectric teleosts need two Nav channel paralogs, how do electric fish
cope with only a single channel?
Muscles have diversified in 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., 1993). It would be intriguing
to know whether Nav channels show a similar pattern of compartmen-
talized expression and rapid evolutionary change in specialized muscles
and muscle-derived organs of these lineages. Has the duplication of a
muscle-expressing Nav channel gene facilitated the evolution of multiple
novel muscle-derived structures in teleosts?
ADAPTIVE EVOLUTION OF NAV CHANNELS:
TETRODOTOXIN RESISTANCE
The best-studied cases of adaptive evolution of Nav channel genes
involve the evolution of resistance to the various neurotoxins that act on
Nav channels. A number of animals use the neurotoxin tetrodotoxin (TTX),
mainly for protection against predators (Gladstone, 1987) but in a few cases
as a weapon to subdue prey (Ritson-Williams et al., 2006). Animals asso-
ciated with TTX span the animal kingdom. This is because TTX is likely
produced by bacteria symbiotically associated with their hosts, or else
taken up from the food chain by animals that prey on TTX-accumulating
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Adaptive Evolution of Voltage-Gated Sodium Channels / 33
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 and Caldwell, 2009). Thus, those animals 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 substitu-
tion. However, TTX resistance in vertebrates is more complex because
vertebrates have multiple Nav channel genes. Evolution of TTX resistance
in vertebrates offers an interesting case of parallel molecular evolution.
Pufferfishes, the most famous being the culinary delicacy Fugu of
Japan, 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 lineages of pufferfishes (Yotsu-Yamashita et al.,
2000; Venkatesh et al., 2005; Jost et al., 2008). We still do not know how
pufferfish were able to survive with only one or a few TTX-resistant Nav
channels. The most likely scenario is that TTX-resistant mutations accu-
mulated gradually in the Nav channel genes as fish were initially exposed
to a light load of TTX. Gradually, as more channels gained resistance, they
were able to carry a greater toxic load. This is suggested by 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
against 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
the 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 extent of TTX resistance in different garter
snake populations suggests that each population has evolved resistance
independently. Even more striking, TTX resistance has evolved multiple
times in populations of other species of garter snakes that are also sym-
patric 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). Finally, sequencing
and testing of expressed Nav channels (Nav1.4, a muscle-expressing Nav
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34 / Harold H. Zakon
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 also have evolved TTX resistance to keep the newts from poisoning
themselves (Kaneko et al., 1997).
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 cannibalistic. 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 nanomolar
levels of TTX that diffuse into the water from the TTX that females place
in their eggs (Matsumura, 1995)]. It is not known yet what receptor detects
the TTX in either newts or pufferfish. One possibility is that it is a Nav
channel that has evolved to open, rather than close, upon TTX binding.
Newt eggs are protected from 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 invertebrates 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.
ADAPTIVE EVOLUTION OF NAV CHANNELS:
PROTON INSENSITIVITY
Naked mole rats (Heterocephalus glaber) live at high density in subter-
ranean 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). The levels of CO2 that build up in 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 examina-
tion of the naked mole rat’s acid-sensing (ASIC) and transient receptor V1
(TRPV1) channels, the channels in vertebrates that subserve acid sensitivity,
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 sensitive
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).
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Adaptive Evolution of Voltage-Gated Sodium Channels / 35
ADAPTIVE EVOLUTION OF NAV CHANNELS IN
REAL TIME: INSECTICIDE RESISTANCE
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., 2000; Davies et al., 2007;
Jones et al., 2012). Starting with their use in the 1940s, the first indications
of resistance, so-called knockdown resistance because insects were no
longer knocked down by normal concentrations 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) from occluding the inner mouth
of the channel. This allows Na+ ions to continue flowing into the cell,
causing hyperexcitabiity. Amino acid substitutions have been discovered
in a variety of insects at a number of sites in the inner mouth of the insect
Nav channel (para in Drosophila) that either reduce pesticide binding or
alter the channel properties 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 chan-
nels counteracting the prolonged channel opening caused by insecticides.
The rapid evolution of Nav channels in insects exposed to insecticides
is one of many warnings we have about the robust abilities of insect pests
to overcome our best attempts to wipe them out.
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 choanofla-
gellates and early metazoans were permeable to both Na+ and Ca2+ and
evolved enhanced selectivity to Na+ in parallel in early bilaterians and jel-
lyfish. Although it is convenient to think that invertebrates possess 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 Devonian
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 sav-
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36 / Harold H. Zakon
ings. It will be intriguing to study the locations and types of Nav channels
in lungfish, basal ray-fin fishes (e.g., bichirs, gars), a variety of tetrapods,
and teleosts to know whether there is parallel evolution of different chan-
nel “types” in teleosts and tetrapods. For example, fast-firing inhibitory
neurons in mammals express different Nav channels than more slowly fir-
ing pyramidal neurons. Do we see a similar functional partitioning of Nav
channel types in teleosts? Are those groups with only four Nav channel
genes (elasmobranchs, basal actinpoterygian fishes, basal sarcopterygian
fishes) hampered in the complexity of their neural processing?
Finally, on a microevolutionary level, we see that Nav channels can be
targets of adaptive changes for increasing diversity in signaling (electric
fish), in the arms race against lethal naturally occurring or synthetic tox-
ins (snakes, newts, pufferfish, insects), and in specialized habitats (naked
mole rats). There are likely to be more examples of this, especially in
animals with unique life histories, and we should keep an eye out for
potentially interesting subjects.
ACKNOWLEDGMENTS
I thank Francisco Ayala, John Avise, and Georg Striedter for organizing
a stimulating Arthur M. Sackler Colloquium of the National Academy of
Sciences and for their invitation to participate. Thank you also to Francisco
Ayala for facilitating the dinner conversation with excellent wines from
his vineyards. Much of the work from my laboratory discussed in this
article was funded by National Institutes of Health Grant R01 NS025513.
