Physiol Genomics 22: 269 –282, 2005.
First published May 24, 2005; 10.1152/physiolgenomics.00229.2004.
CALL FOR PAPERS
Comparative Genomics
Comprehensive analysis of the ascidian genome reveals novel insights into
the molecular evolution of ion channel genes
Yasushi Okamura,1,2,6,8 Atsuo Nishino,1 Yoshimichi Murata,1,2 Koichi Nakajo,2 Hirohide Iwasaki,1,2
Yukio Ohtsuka,6 Motoko Tanaka-Kunishima,4 Nobuyuki Takahashi,2 Yuji Hara,1 Takashi Yoshida,1
Motohiro Nishida,1 Haruo Okado,7 Hirofumi Watari,1 Ian A. Meinertzhagen,5 Nori Satoh,3
Kunitaro Takahashi,4 Yutaka Satou,3 Yasunobu Okada,2,8 and Yasuo Mori1,2,8
Section of Developmental Neurophysiology, Okazaki Institute for Integrative Bioscience, National Institutes of Natural
Sciences, Okazaki, Aichi; 2National Institutes for Physiological Sciences, National Institutes of Natural Sciences, Okazaki,
Aichi; 3Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto; 4Department of Medical
Physiology, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan; and 5Life Sciences Centre, Dalhousie University,
Halifax, Nova Scotia, Canada; and 6Neuroscience Research Institute, National Institute of Advanced Industrial Science and
Technology, Tsukuba, Ibaraki; 7Department of Neurobiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo;
8
School of Life Science, Graduate University for Advanced Studies, Okazaki, Aichi, Japan
Submitted 4 October 2004; accepted in final form 13 May 2005
Okamura, Yasushi, Atsuo Nishino, Yoshimichi Murata, Koichi
Nakajo, Hirohide Iwasaki, Yukio Ohtsuka, Motoko Tanaka-Kunishima, Nobuyuki Takahashi, Yuji Hara, Takashi Yoshida, Motohiro Nishida, Haruo Okado, Hirofumi Watari, Ian A. Meinertzhagen, Nori Satoh, Kunitaro Takahashi, Yutaka Satou, Yasunobu Okada, and Yasuo Mori. Comprehensive analysis of the
ascidian genome reveals novel insights into the molecular evolution of
ion channel genes. Physiol Genomics 22: 269 –282, 2005. First
published May 24, 2005; 10.1152/physiolgenomics.00229.2004.—
Ion fluxes through membrane ion channels play crucial roles both
in neuronal signaling and the homeostatic control of body electrolytes. Despite our knowledge about the respective ion channels,
just how diversification of ion channel genes underlies adaptation
of animals to the physical environment remains unknown. Here we
systematically survey up to 160 putative ion channel genes in the
genome of Ciona intestinalis and compare them with corresponding gene sets from the genomes of the nematode Chaenorhabditis
elegans, the fruit fly Drosophila melanogaster, and the more
closely related genomes of vertebrates. Ciona has a set of so-called
“prototype” genes for ion channels regulating neuronal excitability, or for neurotransmitter receptors, suggesting that genes responsible for neuronal signaling in mammals appear to have diversified
mainly via gene duplications of the more restricted members of
ancestral genomes before the ascidian/vertebrate divergence. Most
genes responsible for modulation of neuronal excitability and pain
sensation are absent from the ascidian genome, suggesting that
these genes arose after the divergence of urochordates. In contrast,
the divergent genes encoding connexins, transient receptor potential-related channels and chloride channels, channels involved
rather in homeostatic control, indicate gene duplication events
unique to the ascidian lineage. Because several invertebrate-unique
channel genes exist in Ciona genome, the crown group of extant
vertebrates not only acquired novel channel genes via gene/genome duplications but also discarded some ancient genes that have
persisted in invertebrates. Such genome-wide information of ion
channel genes in basal chordates enables us to begin correlating the
innovation and remodeling of genes with the adaptation of more
recent chordates to their physical environment.
ascidian; homeostasis; embryogenesis
ION CHANNELS,
membrane proteins that regulate ion fluxes,
provide the molecular bases for diverse physiological phenomena, including neuron function, respiration, absorption, secretion, and the osmotic and volume regulation of body fluids.
Intensive studies in the last two decades have led to the
identification of the molecular nature and detailed biophysical
mechanisms of a wide variety of ion channels. However, there
still remains a large gap between the molecular properties of
ion channels and their macroscopic physiological functions.
Moreover, little is known about the evolutionary history of ion
channel genes and how these may have become remodeled in
the chordate lineage before assuming their vertebrate functions.
Adult ascidians are marine sessile invertebrates, but the
initial stage in their metamorphic life cycle, the tadpole larva,
has a body form with many deep similarities to that of vertebrates. The larval nervous system in particular is both dorsal
and tubular, like the vertebrate neural tube, and similarly shows
an inductive mode of formation during its development (42).
The central nervous system (CNS) of the ascidian larva has
been claimed to contain ⬍100 neurons (48), the cell-lineage
and positions of which have recently been precisely mapped
(9). Later in its life cycle, providing an unusual biological
feature that is characteristic of ascidians, the larva undergoes a
radical metamorphosis to change its body plan and its behavior, transforming in a short period from a motile swimming
form to a sessile filter feeder. In the process, it acquires many
adult-specific organs, such as the heart, pharyngeal gill slits,
and endostyle (16), that are thought also to reflect the evolutionary origins of vertebrate organs. The developmental programs for these organs are proposed to have become decoupled
Article published online before print. See web site for date of publication
(http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: Y. Okamura, Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5-1, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan (email: [email protected]).
1094-8341/05 $8.00 Copyright © 2005 the American Physiological Society
269
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1
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CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
MATERIALS AND METHODS
Retrieving Sequences from the C. intestinalis Genome
and a cDNA/EST Database
The C. intestinalis protein sequences were tblastn searched against
the draft genome sequence [Ref. 11 and DOE Joint Genome Institute
(JGI) Ciona intestinalis v1.0 (JGI site for the complete C. intestinalis
genome sequence and gene annotations): http://genome.jgi-psf.org/
ciona4/ciona4.home.html] and a cDNA/EST database (Ref. 58 and
Ghost Database: http://ghost.zool.kyoto-u.ac.jp/indexr1.html) using human, Drosophila, and C. elegans channel proteins (38). Channel proteins were identified using the following basic method (57).
Briefly, when the corresponding cDNA sequence covering the diagnostic sequences for an ion channel molecule (such as a channel pore
region or a transmembrane region) was available by InterPro search,
the deduced protein sequence was used for the analyses. In surveying
auxiliary subunits for ion channels, the protein sequence was analyzed
when it showed significant overall homology with previously known
Physiol Genomics • VOL
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subunits in the blast search. When the cDNA sequence was not
available and grailEXP or genewise confidently predicted the gene
encompassing the entire channel region, the peptide sequence deduced
from the gene model was used. When the predicted gene model was
not perfect, but the ESTs either covered the entire region or the region
that the gene model lacked, the peptide sequence was deduced from
the assembled sequence obtained using either a set of ESTs (5⬘ and 3⬘
EST pair), multiple sets of ESTs, or both an EST and the gene model.
All analyzed Ciona genes are listed in Supplemental Table S1 (available at the Physiological Genomics web site).1
Molecular Phylogenetic Analysis
The sequences were aligned using the CLUSTALX 1.83 program
(18). We also used another program, MUSCLE (12), which is based
on the algorism with smaller possibility of unreliable alignment
between distantly related sequences. Both alignment programs gave
similar results. Alignments in nonconserved extracellular regions of
channel proteins were carefully checked by eye, and regions with
ambiguous alignments, in particular, at less conserved cytoplasmic
region and extracellular region, were eliminated. Thus verified alignments were used to construct phylogenetic trees both through the
neighbor-joining (NJ) method and maximum-likelihood (ML) analysis for the molecules depicted in Figs. 1–3. For the NJ method, the
trees were calculated using the MEGA program (55). The same fasta
files were analyzed with the ML method using TREE-PUZZLE 5.0
(59). For the analyses shown in Figs. 1–3, the NJ and ML trees were
compared, and branch points were taken significant only when both
analyses gave significant values ⬎50%. Trees of ML analysis following sequence alignment using Clustal program are shown as Supplemental Figs. S4 –S6. For the molecules depicted in Supplemental Figs.
S1–S3, only NJ analysis was performed. The sequences used are
designated in succession by: the accession number, the abbreviation of
the species, and the gene name. Abbreviations of the species are as
follows: HS for human, DM for D. melanogaster, CE for C. elegans,
GG for Gallus gallus, AC for Aplysia californica, EE for Electrophorus electricus, LB for Loligo bleekeri, HR for Halocynthia roretzi, AG
for Anopheles gambiae, HC for Haemonchus contortus, DR for Danio
rerio, XL for Xenopus lavies, CC for Cyanea capillata, BT for Bos
taurus, SP for Strongylocentrotus purpuratus, OB for Oceanobacillus, BH for Bacillus halodurans, AT for Arabidopsis thaliana, and
Hal for Halobacterium sp. NRC-1.
Best-Hit Gene Analysis
We compared the indicated Ciona proteins with the International
Protein Index human proteome. At first, identified proteins were
compared using the blastp program (1). The best-hit protein in the
human proteome was then blastp searched against the Ciona version
1 proteome without the option of gapped alignment. When the best-hit
sequence of the human protein corresponded to the region encoding
the starting Ciona protein, the relationship between the two proteins
was called the “bidirectional best-hit relationship.” Otherwise, it was
called a “unidirectional best-hit relationship.” To define the orthology
between the two proteins, either bidirectional best-hit relationship or
a bootstrap value of ⬎50% in both NJ and ML methods was taken as
the criteria.
RESULTS
A survey of the C. intestinalis genome identified 160 genes
with homology to ion channels. Ion channel-like genes were
grouped into several classes: voltage sensor-containing cation
1
The Supplemental Material for this article (Supplemental Table S1 and
Supplemental Figs. S1–S6) is available online at http://physiolgenomics.
physiology.org/cgi/content/full/00229.2004/DC1.
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during evolution and in current forms to be represented at
different stages of the ascidian life cycle, in the larva and adult
(20).
Recently, the entire genome of one species of ascidian,
Ciona intestinalis, has been sequenced (11). The draft Ciona
genome contains ⬃16,000 protein-coding genes, a number
which is intermediate between those of protostome and vertebrate genomes. More than 10% of the genes are specific to
chordates, whereas 63% are common to both chordates and
protostomes. The Ciona genome is ⬍160 Mb, which is more
compact and dense (7.5 kb/gene) than the genomes of either
Drosophila (9 kb/gene) or the human (100 kb/gene). This
recommends ascidians as an excellent model to reveal gene
networks by systematic analysis of promoters. Combined with
the wealth of information recently obtained about gene expression profiles, provided especially from expressed sequence tag
(EST) [Ref. 58 and Ghost Database (a C. intestinalis cDNA
resource): http://ghost.zool.kyoto-u.ac.jp/indexr1.html] and
in situ hybridization data (50), several groups of genes from the
Ciona genome have been surveyed systematically, and their
relations with vertebrate homologs have been analyzed. These
genes include those related to the cytoskeleton, muscle contraction, signal transduction, cell interactions, and transcription
(57). With the background provided by findings from classical
physiology (16), when combined with this recent flood of
genomic information, ascidians provide a unique opportunity
to gain insights into the origin of vertebrate physiological
functions.
Here we have systematically surveyed the putative ion
channel genes in the genome of C. intestinalis and compared
them with the comparable gene sets of Chaenorhabditis elegans, Drosophila melanogaster, and the human genomes. In
addition, we have also referred to the genome of the Japanese
puffer fish Fugu rubripes for some ion channel genes. Not
surprisingly, we find that the Ciona genome contains a minimum set of prototype genes for neuronal signaling, voltagegated cation channels and transmitter-gated ion channels.
Channel genes related on the other hand to homeostatic control, such as transient receptor potential (TRP) and connexins,
comprise not only vertebrate prototype genes but also multiple
genes that are more specialized and belong to ascidian-specific
clades. Ion channel genes for fine-tuning of excitability and
pain-related channels are not present in the Ciona genome.
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
channels, transmitter-gated channels, anion channels, non-voltage-gated cation channels, intracellular calcium channels, and
intercellular channels. Channel gene orthologs were identified
as genes that showed bootstrap values ⬎50% at the diagnostic
branch points in the phylogenetic trees or, alternatively, a
bidirectional best-hit relationship in the best-hit gene analysis
(see also MATERIALS AND METHODS).
Voltage Sensor-Containing Cation Channels
TuNa2, share two unusual features in their primary structure:
first, the lysine residue critical for sodium selectivity in the
pore region of the domain III is replaced by glutamic acid in
TuNa2; and the amino acid sequences of the III-IV linker, a
region known to function as the inactivation ball (19), are not
well conserved. These two features of the primary structure are
shared by other Nav-like genes from three species of protostome: Drosophila, the cockroach, and squid. Recent biophysical studies have shown that, in the cockroach, the gene of this
invertebrate-specific clade encodes a slowly inactivating calcium-permeable channel, suggesting that Ci-Nav2 could be
permeable to both sodium and calcium (70). The other two Nav
channels (Ci-Nav3, Ci-Nav4) are related to each other and
located at a tree position that is intermediate between those for
the protostome and vertebrate branches of the molecular phylogeny (Fig. 1A).
Ciona has single genes for the three main subclasses of
voltage-gated calcium (Cav) channels: Ci-Cav1, Ci-Cav2, and
Ci-Cav3 (Fig. 1B). The presence of representatives from all
three channel types is consistent with a previous view that the
diversification of these three predated the separation of deuterostome and protostome animals (38) (Supplemental Fig.
S1). Because there are multiple paralogous genes belonging to
each of the subfamilies of Cav1–3 in the Fugu genome and the
Fig. 1. Phylogenetic trees by the neighbor-joining (NJ) method for voltage sensor-containing channels. A: Nav channel ␣-subunit. B: Cav channel ␣1-subunit.
C: Kv1– 4 channel ␣-subunit. D: KCNQ channel ␣-subunit. E: cyclic nucleotide-sensitive cation channel genes. Branch points supported by both the NJ and
maximum-likelihood (ML) methods are shown by red circles. Bootstrap values of NJ and ML analyses are indicated in black and red letters, respectively.
Physiol Genomics • VOL
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Main channel-forming subunits. Four putative genes coding
for ␣-subunit proteins of voltage-gated sodium, Nav, channels
exist in the Ciona genome (Fig. 1A). One of these (Ci-Nav1) is
a counterpart of TuNa1, a neuronal Nav channel gene previously characterized in another species of ascidian, H. roretzi
(53). This gene is most closely related to the group of vertebrate Nav channels. The other three genes (Ci-Nav2, Ci-Nav3,
Ci-Nav4) are only distantly related to the authentic chordate
branch of the Nav channels. One of the three nonconventional
Nav channels (Ci-Nav2) shows a high level of sequence
similarity to the ascidian cation channel, TuNa2, which is
predominantly expressed in Halocynthia larval motor neurons
(46). Its cDNA expression profile (58) indicates that this gene
is expressed in both the nervous system and endostyle in the
adult ascidian. The products of both genes, Ci-Nav2 and
271
272
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Table 1. continued
Table 1. Comparison of nos. of ion channel subunit genes
among Drosophila, C. elegans, C. intestinalis, and H. sapiens
C. elegans
Ciona
H. sapiens
2
0
2
0
0
0
4
0
0
11
4
0
1
1
1
1
1
0
3
0
0
1
1
1
2
2
0
3
0
0
1
1
1
1
1
1
2
3
2
4
4
3
1
4
8
4
3
2
1
1
2
1
0
0
1
1
1
0
0
0
0
1
0
3
1
0
6
3
2
2
0
0
0
0
3
2
0
1
0
0
2
1
1
2
1
0
0
8
2
4
3
8
0
5
1
1
4
4
1
4
1
6
1
1
1
0
0
4
1
0
1
0
3
3
1
1
1
5
4
6
3
3
2
0
30
15
3
1
1
0
4
6
15
4
3
1
1
0
4
2
11
1
1
1
1
2
2
3
17
5
4
1
5
0
2
0
10
0
1
10
0
0
0
1
2
0
7
1
0
13
1
6
3
5
42
0
1
37
1
0
2
2
1
0
31
1
0
11
4
2
5
3
8
3
1
7
1
3
2
0
0
1
0
3
0
27
2
8
8
9
11
2
2
23
11
3
6
0
0
3
0
1
7
25
8
7
6
7
24
2
0
22
22
0
0
22
7
2
0
5
9
5
4
0
Physiol Genomics • VOL
Cl⫺ channels
CLCN
CLCA
CLIC
CFTR
Ca2⫹ release channels and
intercellular channels
IP3R
RyR
connexin
Mip/AQP
Non-voltage-gated K⫹ channels
K2P
IRK
GIRK
ATP-sensitive
others
SK
IK
Drosophila
C. elegans
Ciona
H. sapiens
4
3
0
1
0
7
6
0
1
0
13
5
7
1
0
21
9
5
6
1
1
1
0
2
1
1
0
5
1
1
17
6
3
3
22
11
11
3
0
0
0
1
0
50
3
0
0
0
4
0
5
1
2
0
1
1
0
10
4
4
2
5
3
1
Subunit, not composing ion channel pore, is indicated in italic. C. elegans,
Chaenorhabditis elegans; C. intestinalis, Ciona intestinalis; H. sapians, Homo
sapiens.
human genome [Fig. 1B, Supplemental Fig. S1, and JGI Fugu
rubripes v3.0 (JGI Fugu genome site): http://genome.jgi-psf.org/
fugu6/fugu6.home.html], duplications within each Cav subfamily (for example, into Cav1.1, Cav1.2, Cav1.3, Cav1.4)
must have been established somewhere between the branch
points of the urochordate and the shared ancestor of teleosts
and mammals. In addition to these three prototype Cav channel
genes, Ciona has several genes that are homologous to mammalian “Cav-like channels,” the functions of which remain to
be determined. One is the four domain-type channel (35)
recently called ␣1U, which is highly conserved in the Drosophila,
C. elegans, and vertebrate genomes. Two other genes, Ci-TPC1
and Ci-TPC2, putatively encode the two-domain type channel,
which has been reported in the human genome but in neither the
Drosophila nor C. elegans genomes (Supplemental Fig. S2).
Ciona has three genes closely related to the one-domain type of
voltage-gated cation channel, CatSper, which is known to play a
role in sperm motility (8) (Supplemental Fig. S2).
A minimum set of genes also seems to be present for
voltage-gated potassium channel (Kv) proteins (Fig. 1C).
Ciona has three Kv1, one Kv2, one Kv4, one gene distantly
related to all Kv channels, and two KCNQ genes (Fig. 1D).
Unexpected position of human Kv4 at the base outside the
clade of Kv4-related genes (Fig. 1C) was not supported when
only a group of Kv4-related genes was aligned independently
(data not shown). No Kv3-like gene was found in the Ciona
genome. Ciona has genes for the calcium-activated BK, Slack,
and SK potassium channels (Supplemental Fig. S2, Table 1).
The CNG-ch, HCN-ch, and eag/erg/elk channel genes all
belong to a large family of genes putatively encoding sixtransmembrane cation channel proteins that contain an S4-like
voltage-sensor region and a nucleotide-binding region at their
COOH terminus (19). Ciona has a single gene for each of the
erg, elk, and eag subfamilies (Ci-erg, Ci-elk, Ci-eag). The
CNG channel genes number three in the Ciona genome compared with four in the Drosophila and six in the C. elegans
genome (Table 1, Fig. 1E, Ci-CNG1 to -3). There are three
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Voltage-gated Na⫹ channels
alpha
beta
TipE
Ca2⫹ channels
alpha(Cav1)
alpha(Cav2)
alpha(Cav3)
alpha(novel)
beta
gamma
alpha2/delta
1-domain Cav-like channel
2-domain Cav-like channel
Voltage-gated K⫹ channels
Kv1
Kv2
Kv3
Kv4
Kv5,6,8,9
divergent Kv
KCNQ
BK(KCa1.1)
Slack(KCa4.1)
BK beta
Kv beta
KCNQE-minK
KCNQE-MirP
Cyclic nucleotide-sensitive cation
channels
Ih (HCN)
CNG
erg-related
elk-related
eag-related
other erg/elk/eag
Glutamate receptor channels
total iGluR
KainateR
AMPAR
NMDA-R1
NMDA-R2, R3
NMDA-non R1/2/3
deltaR
others
ACh receptor channels
AChR
5HT3R
rapsyn
GABAR/GlyR channels
GABA-alpha/epsilon/gamma
GABA-rho
GABA-beta/pi/delta
GluCl
histamine-gated Cl Ch
GlyR
others
gephyrin
P2X receptor channels
TRP channels
TRPM
TRPC
TRPV/N/A
PKD (TRPP)
Amiloride-sensitive cation
channels
ASIC
ENaC
others
Drosophila
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Transmitter-Gated Ion Channels
GABA, glycine, glutamate, and acetylcholine (ACh) are all
common chemical neurotransmitters in both vertebrate and
invertebrate nervous systems. Despite the commonality of their
neurotransmitter ligands, previous studies suggest that vertebrates and invertebrates do not necessarily share all forms of
neurotransmitter receptors. For example, C. elegans and Drosophila have both histamine- and glutamate-gated Cl⫺ channels, but they lack G protein-coupled receptors, which are
present in vertebrates (54). Moreover, vertebrates have serotonin-gated cation channels, glycine receptors, and ATP-gated
channels that do not exist in the genomes of C. elegans and
Drosophila.
Nicotinic ACh receptor family. Two types of subunits of
nicotinic ACh (nACh) receptors (nAChR) are known: ␣-subunits, with the ability to bind ACh, and non-␣-subunits, which
lack such binding (63). Ciona has four nAChR ␣-like genes
and four non-␣-subunit genes (Fig. 2A). All four ␣-like genes
show two neighboring cysteines in the extracellular or C-loop,
which is essential for ACh-binding activity (63). One Ciona
␣-like gene, Ci-nAChR-A1, clusters with the ␣-subunit genes
of the nAChR receptor of vertebrate skeletal muscle in the
Physiol Genomics • VOL
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molecular phylogenetic tree (Fig. 2A). This is consistent with
the finding that ascidian larval muscle is known to express
cation-permeable cholinergic receptors (51), resembling pharmacologically those at vertebrate skeletal muscle. Two Ciona
␣-like genes, Ci-nAChR-A7/8-1 and -2, cluster with the ␣7and ␣8-subunit mammal genes, which are bungarotoxin-sensitive neuronal nAChR receptors, and with the nAChR receptor
genes of C. elegans and Drosophila (Fig. 2A). This indicates
that the bifurcation between neuronal and skeletal muscle-type
␣-nAChR subunit genes occurred early, before urochordates
diverged from the other chordates. Ciona has a gene closely
related to the ␣3-subunit, Ci-nAChR-A3, that is present in the
genomes of neither Drosophila nor C. elegans. All four non␣-subunit genes in the Ciona genome lack the two neighboring
cysteines in the C-loop region for ACh binding, consistent with
the idea that they must coassemble with ␣-subunits to be
functional. One gene is closely related to the ␤2- and ␤4subunit genes of vertebrates, designated Ci-nAChR-B2/4, while
three non-␣-subunit genes are grouped just outside the non-␣subunit genes of vertebrate skeletal muscle nAChR (Fig. 2A,
Ci-nAChR-B/G/D/E1 to -3). The pentameric nAChRs of vertebrate skeletal muscle contain four non-␣-AChR subunits (␤,
␥, ␦, and ε) (19). Without evidence of gene expression in the
ascidian muscle lineage cells, it would be premature to conclude that the three Ciona non-␣-genes are orthologs of vertebrate non-␣-skeletal muscle subunits, but it is apparent that the
divergence of these Ciona non-␣-subunit genes (Ci-nAChR-B/
G/D/E1–3) might have occurred independently of diversification of the non-␣-subunits for vertebrate skeletal muscle.
In agreement with the presence of ␣-like nAChR subunit
genes and the cholinergic nature of neuromuscular transmission (51), Ciona also has a single gene homologous to rapsyn,
which is involved in clustering ACh receptors in postsynaptic
domains of vertebrate skeletal muscle (Table 1).
The 5HT3 receptor is a serotonin-gated receptor channel
with significant homology to nAChRs that hitherto has been
found only in vertebrates. The Ciona genome has three genes
related to the vertebrate 5HT3 receptor genes, and they constitute a single clade within the AChR/5HT3R tree (Fig. 2A).
Ionotropic glutamate receptor family. The Ciona genome
has single prototype genes for each of the AMPA receptor
(Ci-GluR1/2/3/4) and N-methyl-D-aspartate (NMDA) receptor
(Ci-GluR-NR1 and -NR2) types of ionotropic glutamate receptor (GluR), as supported both by the clear bidirectional best-hit
relationship and by their high bootstrap values (Fig. 2B). There
is one kainate receptor-like gene (Ci-GluR-KaiR-like). However, this similarity to kainite receptor gene was not significantly supported by the ML method. Ciona has two genes
related to the ␦-subunit subfamily, Ci-GluR-Delta1 and -2.
Ci-GluR-Delta2 showed a bidirectional best-hit relationship,
whereas Ci-GluR-Delta1 showed a unidirectional best-hit relationship. The presence of ␦2-like subunit gene in Ciona was
unexpected, because the ␦2-subunit is expressed predominantly in cerebellar Purkinje neurons in mammals (39),
whereas no structural counterpart of the cerebellum has been
identified in the ascidian nervous system.
A remarkable feature of vertebrate glutamate receptors is
that the critical site for Ca2⫹ permeability of several of the
GluR subtypes is subject to RNA editing, which has been most
intensively characterized in the AMPA receptor genes. Conversion through RNA editing of Q to R in the M2 domain
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HCN-like genes in the Ciona genome, whereas there is only a
single gene in the Drosophila genome and none in C. elegans
(Table 1, Fig. 1E). The Ciona genome also has four other genes
(Ci-eag/HCN/CNG-diverged1– 4 distantly related to known
CNG/HCN/eag, erg, elk channels) (Fig. 1E).
Auxiliary subunits. Auxiliary subunits modify the gating
kinetics, localization, and expression levels of the ␣-subunits
of voltage-gated channels and are therefore functionally important modifiers of electrical excitability. The Ciona genome
contains ␤-, ␥-, and ␣2/␦-subunit genes for Cav channels
(Supplemental Fig. S2). For auxiliary subunits at voltage-gated
potassium channels, Ciona has a single Kv ␤-subunit-like gene
and two genes putatively encoding BK channel ␤-subunits
(Table 1 and Supplemental Fig. S2), and at least one putative
KChip gene encoding a calcium-binding auxiliary subunit for
Kv4-class channel proteins (Ref. 2 and data not shown). These
genes are present in neither the Drosophila nor the C. elegans
genomes. By contrast, Ciona lacks many other auxiliary subunit genes. Vertebrate Nav ␤-subunits support the clustering of
Nav channel proteins at the node of Ranvier and initial segments of vertebrate myelinated nerve fibers, through interactions with myelin-forming glial cells. The absence of a Nav
␤-subunit gene in the Ciona genome (Table 1) is therefore
consistent with the lack of both myelin-related genes in the
genome (11) and myelin-like structures in ascidian nerves (30).
The Ciona genome also lacks a TipE gene, which is known to
substitute for the role of Nav-␤ in Drosophila (21). The Ciona
genome also lacks two other classes of auxiliary subunits for
potassium channels: first, KCNQE genes including Mirp and
MiniK genes for fine-tuning the gating of KCNQ-type potassium and erg channels; and, second, genes for Kv5,6,8,9
subunits, which are known to modulate the function of Kv2
channels (Table 1).
To summarize, Ciona has a simple set of prototype gene
homologs similar to those for vertebrate voltage-gated channels but lacks the Kv3 gene and most of the auxiliary subunit
genes.
273
274
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
occurs in transcripts of vertebrate GluR subunits and eliminates
the Ca2⫹ permeability of the receptor (60). The genome sequence of the putative AMPA receptor-like gene of Ciona
shows that the site corresponding to the Q/R site of the
mammalian AMPA receptors is also Q. Detailed comparison
between the genome and cDNA sequences will clarify whether
RNA editing also underlies conversion of the Ca2⫹ permeability of GluR channels in the urochordate.
In addition to the above GluR genes, Ciona has three other
GluR-like genes with lower homology to vertebrate GluR
channel genes, and these form a Ciona-specific clade (Fig. 2B;
Ci-GluR-Div1 to -3). This separate ascidian gene group suggests that gene diversity among ascidian GluR-like genes was
established independently of the diversification of GluR genes
in vertebrate evolution, i.e., that it occurred after the urochordate/vertebrate split and that the counterparts were lost in the
vertebrate lineage. Two other Ciona genes (Ci-GluR-Div4 and
-5) seem distantly related to vertebrate NMDA receptor genes,
although this relationship was not supported by the ML tree
(Fig. 2B).
Finally, Ciona has two genes putatively encoding a GRIPlike protein and PSD-95, which are involved in the scaffolding
of GluR at the postsynaptic membrane and which play roles in
the processing of synaptic plasticity (data not shown).
Physiol Genomics • VOL
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Ionotropic GABA/glycine receptor family. There are in total
eight ionotropic GABA/glycine receptor (iGABA/GlyR) genes
in the Ciona genome, including one GlyR-related gene and
seven others, all GABAR-like genes; the seven include one
␣/␥/ε-related gene, one ␲-related gene, one ␤-related gene, and
four ␳-related genes (Table 1, Fig. 2C). The presence of
multiple subfamilies of genes for iGABA/GlyR genes in the
Ciona genome agrees well with the gene diversities in Drosophila and C. elegans, suggesting that main diversifications of
the iGABA/GlyR receptors predated the deuterostome/protostome bifurcation and also implying that GABA and glycine
may both have acted as signaling molecules in basal metazoan
ancestors. However, there are two remarkably distinct aspects
of the Ciona genes that distinguish them from the iGABA/
GlyR genes of Drosophila and C. elegans. First, one Ciona
gene, Ci-GlyR, is grouped with vertebrate GlyR genes but with
neither the genes for glutamate-gated Cl⫺ channels (GluCl) nor
histamine-gated Cl⫺ channels, both of which are so far unidentified in vertebrates (Fig. 2C). Second, the Ciona genome
contains four ␳-related subunits (Ci-GABAAR ␳1 to -4), subfamilies that are not found in the Drosophila and C. elegans
genomes. All eight iGABA/GlyR genes putatively encode
Cl⫺-permeable channels, since their sequences encode conserved amino acids of the pore-forming region, including the
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Fig. 2. Phylogenetic trees by the NJ method for transmitter-gated channels. A: ACh receptor (AChR) genes. B: glutamate receptor (GluR) genes. C: GABAA
receptor (GABAAR)/glycine receptor (GlyR)-related genes.
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Non-Voltage-Gated Cation and Anion Channels
Inwardly rectifying K⫹ channel and two-pore domain K⫹
channels. Inwardly rectifying K⫹ channel (Kir) and two-pore
domain K⫹ channel (K2P) genes encode important channels
for determining the resting membrane potential and for the
transport of potassium ions. Ciona has four Kir-like genes,
including one gene (Ci-Kir2/5) related to the classical Kir
subfamily (Kir2), two genes (Ci-GIRKA, Ci-GIRKB) related to
Kir3 (GIRK), which is activated by a G protein, and one gene
(Ci-Kir1/4/7) loosely related to subfamily of Kir1/4/7 (Table 1,
Supplemental Fig. S3). Such diversity is found neither in C.
elegans nor in Drosophila (Table 1), suggesting that these
prototypes of the different Kir subfamilies emerged before the
appearance of the urochordate line and probably after the
divergence between protostomes and deuterostomes. On the
other hand, the Ciona genome lacks a gene for the ATPsensitive K⫹ channel subfamily, which is known in vertebrates
to regulate excitability as a function of the metabolic state in
muscle, brain, and endocrine cells (Table 1). Accordingly,
there is no homolog in the Ciona genome of the SUR-type
ATP-binding cassette (ABC) transporter gene, which coassembles with ATP-sensitive K⫹ channels in vertebrates.
K2P, the non-voltage-gated K⫹ channel consisting of two
domains, also plays a critical role in determining the resting
membrane potential. The Ciona genome has a total of five
K2P-like genes, Ci-TWIK1 to -5, classified in two subgroups
that are conserved among the genomes of Drosophila, C.
elegans, and vertebrates. These five include four genes in the
TWIK1-related clade and a single gene in the TASK3-containing clade (Supplemental Fig. S3). The number of Ciona twodomain K⫹ channel genes is far fewer than in the genomes of
Drosophila (38) and C. elegans (36, 38) (Table 1).
Physiol Genomics • VOL
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Amiloride-sensitive cation channels. Amiloride-sensitive
cation channels are two-transmembrane sodium-permeable
channels. In mammals, amiloride-sensitive cation channels
consist of two main subfamilies: the acid-sensing ion channel
(ASIC) group, channels expressed in neurons, and the epithelial Na⫹ channel (ENaC) subfamily, abundantly expressed in
epithelia such as in the lung, kidney, and intestine. The Ciona
genome contains two genes that belong to the ASIC branch
(Ci-ASIC/degenerin6,7) and five additional genes (Ci-ASIC/
degenerin1–5)that show only weak homology to amiloridesensitive cation channels (Fig. 3A). One of the five, Ci-ASIC/
degenerin3, showed a bidirectional best-hit relationship with
the human amiloride-sensitive cation channel gene, ACCN5,
supporting the conclusion that it encodes an amiloride-sensitive cation channel. The C. elegans and Drosophila genomes
also show species-specific branches of putative amiloridesensitive cation channel genes, but the five Ciona genes with
weak homology to amiloride-sensitive cation channel genes
lack any close relationship to the genes of the C. elegans and
Drosophila clades, indicating that the diversities for this channel group of deuterostomes and protostomes are derived independently. Although NJ-based tree suggested that these five
genes were weakly related to the vertebrate ENaC genes (Fig.
3A), it is unlikely that Ci-ASIC/degenerin1–5 correspond to the
orthologs of ENaC, since neither alignment with MUSCLE nor
tree formation with the ML method supported this relationship.
The best-hit gene analysis also did not support the orthology of
these genes to the vertebrate ENaC subfamily (Supplemental
Table S1).
TRP-related channels. TRP-related channels are six-transmembrane cation channels that respond to various sensory
modalities, such as temperature sensation, pain sensation, and
osmotic stress (8). Exceeding the number found even in the
human genome, Ciona has a total of 27 TRP-related genes
(Table 1), with representatives covering all four known subfamilies, TRPM, TRPC, TRPV/N/A, and TRPP [or polycystic
kidney disease (PKD)-related channel] (Fig. 3, B–D; see Supplemental Fig. S3). There are two TRPM-like genes, one of
which, Ci-TRPM2/4/8, codes for NUDT9, a Nudix hydrolase
domain corresponding to that found in the mammalian TRPM2
channel (Supplemental Fig. S3). The Ciona genome has a total
of eight TRPC-like genes (see Fig. 3B). One of these is
grouped with vertebrate members, whereas the other seven
genes are located outside the vertebrate branch. The single
gene grouped with vertebrate TRPC genes (Ci-TRPC4/5) codes
for a TRPC-specific TRP box sequence, EWKEAR, at the
COOH terminus. This motif is well conserved in the other
seven TRPC-like genes, although alanine is replaced by H or Y
or Q. Of the seven genes located outside the mammalian TRPC
channel genes, five contain multiple ankyrin repeats at the NH2
terminus, as also do mammalian TRPC channels (Fig. 3B).
The Ciona genome has an unusually large diversity of
PKD-related channel (TRPP) genes, nine in all (Table 1, Fig.
3C). The vertebrate TRPP subfamily consists of three subclasses, PKD1, PKD2, and the mucolipin-related gene, all of
which were first identified as genes responsible for human
genetic disorders. The Ciona genome seems to contain counterparts both for PKD1 and PKD2 (Ci-PKD1 and Ci-PKD2,
respectively) that are known to coassemble to form Ca2⫹permeable channels in the kidney (17) (Fig. 3C). Although
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cytoplasmic rings, intermediate rings, and extracellular rings at
the M1 and M2 region, which are known to determine anion
selectivity (Ref. 28 and data not shown).
Even though there is only a single GlyR-like gene, there are
three genes present in the Ciona genome putatively encoding
gephyrin (Table 1), which induces clustering of GlyR in
vertebrate neurons. The fact that gephyrin genes outnumber the
single GlyR gene in Ciona raises the possibility that ascidian
gephyrins might have roles other than that of regulating the
distribution of glycine receptors.
The Ciona genome does not contain the purinergic receptor
channel P2X (Table 1). Correspondingly, we also could not
find any gene homologous to the G protein-coupled type of
purinergic receptor, P2Y, in the Ciona genome, consistent with
the idea that neither ATP nor UTP are utilized as extracellular
signaling molecules in ascidians.
To summarize, just as for voltage-gated channel genes, the
Ciona genome contains conserved sets of prototype genes for
most of the subfamilies of vertebrate transmitter-gated channels. Overall, the number of transmitter-gated channel genes is
fewer in Ciona than it is in either C. elegans or Drosophila
(Table 1). Our findings also suggest that the basic combinations of transmitter-gated channels found in vertebrates (the
presence of GlyR and 5HT3R but the lack of GluCl and
histamine-gated channels) were already established in ancestral
forms before the divergence of urochordates.
275
276
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
cantly indicated by the ML-based tree following alignment
with CLUSTAL software (Supplemental Fig. 6C), this was
supported by both the bidirectional best-hit analysis and alignment with MUSCLE software followed by NJ-based tree
formation. Ciona has only a single mucolipin-like gene (Cimucolipin), which fits in between its Drosophila and human
homologs, not contradictory to the intermediate position of
ascidians. In addition to these prototype genes, the Ciona
genome contains six other TRPP-related genes, broadly scattered in the phylogenetic tree (Fig. 3C).
The Ciona genome has two TRPV-like genes, but none of
these is grouped with vertebrate members of this family but
rather with the “osm-9 channel” gene of C. elegans which is
required for osmotic/mechanical sensation (Fig. 3D) (45). One
of the two Ciona osm9-related genes, Ci-Osm9-related2, is
likely the counterpart of a previously identified osmosensitive
channel from another ascidian, H. roretzi (32). Ciona has only
a single gene member of the TRPN subfamily, Ci-TRPN,
related to the teleost gene NOMPC, which putatively encodes
a mechanosensory channel in hair cells and neuromast organs
of the lateral line in zebrafish (62) (Fig. 3D). The Ciona
genome has four TRPA-like genes with extensive ankyrinrepeats at the COOH terminus, whereas in mammals there is
only a single TRPA member (ANKTM1, Fig. 3D), which has
Physiol Genomics • VOL
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recently been claimed as a mechanosensory channel in cochlea
(10). It would be interesting to know whether the Ciona TRPV,
TRPN, or TRPA-like genes express in peripheral neurons of
the larval trunk (65) or in ciliated secondary neurons of the
adult coronal organ (7) or other candidate mechanoreceptor
neurons.
Cl⫺ channels. The Ciona genome has a number of genes for
⫺
Cl channels, including the CLIC, CLCN, and CLCA groups
(Fig. 3E; see Supplemental Fig. S3). The Ciona CLCN channel
genes correspond to the mammalian subfamilies of CLCN3/4/5
and CLCN1/2 and CLCN6/7 (Supplemental Fig. S3). The
Ciona genome has seven CLCA genes that exhibit considerable duplications (Fig. 3E), which makes a sharp contrast with
the absence of such homologs in Drosophila or C. elegans
(Table 1). This suggests that the CLCA family is a specific
feature of chordate or deuterostome genomes (Fig. 3E). The
Ciona genome has only a single CLIC-like gene (data not
shown).
There is no cystic fibrosis transmembrane conductance regulator (CFTR)-like gene found among the numerous ABC
transporter genes (Table 1). The Ciona genome also lacks a
gene of the CLC-K subfamily, which is specific to the kidney
in vertebrates (31). Its absence from Ciona is of course compatible with the absence of any organ like the kidney in
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Fig. 3. Phylogenetic trees by the NJ method for non-voltage-gated cation channels and anion channels. A: amiloride-sensitive channel genes. B: TRPC channel
genes. C: polycystic kidney disease (PKD)-related channel genes. D: transient receptor potential (TRP) and TRPN/A channel genes. E: CLCA channel genes.
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Other Channels
Inositol trisphosphate receptor and ryanodine receptors.
The inositol trisphosphate (IP3) receptor and ryanodine receptor channels play important roles in the intracellular release of
Ca2⫹ from internal stores (5), which for example underlies
Ca2⫹ waves in the fertilized Ciona egg (69). There is only a
single gene for each of the IP3 and ryanodine receptor families
in the Ciona genome (Table 1, Supplemental Fig. S3). On the
other hand, Fugu has three IP3 receptor genes, each grouped
with the corresponding mammalian subtype gene (Supplemental Fig. S3), indicating that, like the genes for voltage-gated
channels, the diversity of IP3 receptors and ryanodine receptors
was established during the course of chordate evolution after
the divergence of the urochordate lineage.
Gap junctions and water channels. Connexins serve to
mediate the diffusion of small molecules between cells and
thereby play many critical roles in intercellular communication
during neural development and function (68). As previously
reported (56), the Ciona genome has 17 putative connexin
genes. Although these Ciona genes all belong to the large
family of vertebrate connexins, most are independent of any
subclass of vertebrate connexins in the phylogenetic tree (data
not shown). As pointed out previously (56), Ciona lacks
homologs of the invertebrate innexin gene family and, instead,
has two genes with some homology to the human pannexin
genes, which revealed another clear deuterostome/protostome
distinction (56). Water channels play roles in osmoregulation
and the regulation of cell volume in a variety of species from
plants to mammals. The Ciona genome contains a total of six
aquaporin-like genes, four of these related to human aquaporin-8, namely Ci-AQP8-1 to -3 and Ci-Drip, and one called
Ci-AQP3 that is related to human aquaporin-3/7/9/10 (see
Supplemental Fig. S3). A sixth gene is located at some distance
from the other five genes.
Clustering of Homologous Genes in Tandem
In our survey of ion channel genes, we noticed that multiple
homologous genes are often clustered in restricted regions of
the genome. These include genes for one pair of GABAA-R
genes (Ci-GABAAR-␳1:Ci-GABAAR-␳2; see Supplemental Table S1 of the total list of Ciona channels for gene nos.), four
eag/HCG/CNG-related K⫹ channel genes (Ci-eag/HCN/CNGdiverged4:Ci-eag/HCN/CNG-diverged1:Ci-eag/HCN/CNG-diPhysiol Genomics • VOL
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verged3:Ci-eag/HCN/CNG-diverged2), two pairs of amiloridesensitive channel genes (Ci-ASIC/degenerin6:Ci-ASIC/degenerin7 and Ci-ASIC/degenerin5:Ci-ASIC/degenerin4), a pair
of TRP-C-like channel genes (Ci-TRPC-related6:Ci-TRPCrelated5), two pairs of connexin genes (Ci-connexin-related5:Ci-connexin-related-6 and Ci-connexin-related-11:Ci-connexin-related-4), one pair of nAChR genes (Ci-nAChR-B/G/D/
E2:Ci-nAChR-B/G/D/E3), and one pair of CLCA Cl⫺ channel
genes (Ci-CLCA1:Ci-CLCA3). In the case of the genes for the
eag/HCN/CNG-related K⫹ channels, four K⫹-channel like
genes are aligned next to each other in the same direction in
tandem with an intergenic region of ⬍2 kb. This arrangement
might reflect the origin of four genes from two rounds of
duplications from a single ancestral gene. Because these channel species are known to produce channel functions by heterologous subunit assembly, the clustering of homologous genes
in tandem in the Ciona genome may reflect an operon-like
regulation of a set of genes. The significance of such clustering
of ion channel genes must, however, await extensive examination of expression patterns of the corresponding transcripts
and studies of promoter regions of the individual genes.
Ion Channel Genes Are Expressed During Embryogenesis
Recently, many examples have shown that ion channel
activities play critical roles in the morphogenesis (6) and early
development of embryos (41). We searched the C. intestinalis
EST database of Kyoto University (http://ghost.zool.kyoto-u.
ac.jp/indexr1.html; Ref. 58) for ion channel genes, which are
expressed in early embryos. Eggs and early embryos express
genes putatively encoding the following: TWIK, connexin,
Kir2/5, PKD-related channel, eag/HCN/CNG diverged channel, Cav3-related channel, Cav channel ␣2/␦-, ␤-subunits,
two-domain Ca2⫹ channel (TPC2), KCa ␤-subunit, and voltage-dependent Cl⫺ channels (Table 2). Early expressions of
auxiliary subunits of the gene for Cav channel and Kir2/5 are
compatible with the developmental profiles of ion currents
previously revealed electrophysiologically (64), in which voltage-gated Ca2⫹ channels and inward rectifier K⫹ channels
emerge at early stages.
DISCUSSION
Ciona Has Restricted Sets of Voltage- and TransmitterGated Channels That Are Chordate Prototype Genes
The Ciona genome shows only a rather minimal set of
voltage-gated channels that are presumed to represent ancestral
prototypes of those also inherited by vertebrates. The number
of such genes is roughly as large as those in Drosophila and C.
elegans, except that the Ciona genome has more Kv1-like and
Nav-like genes. These comparisons suggest that diversification
within each subfamily of voltage-gated channels occurred only
after the divergence between urochordates and other chordates.
In fact, even though fish brains are themselves highly diverse
(4), fish genomes already show a pattern of gene diversity for
voltage-gated channels that is as great as that in mammalian
genomes. For example, the Fugu genome contains prototype
genes corresponding to all five of the mammalian genes,
Cav1.1 to Cav1.5 [see Supplemental Fig. S2 and the JGI Fugu
genome site (http://genome.jgi-psf.org/fugu6/fugu6.home.
html)], and electric fish genome contains multiple Nav channel
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ascidians. On the other hand, the Fugu genome also lacks a
CLC-K-like gene as well as an ortholog of the ENaC gene
(data not shown), even though this fish clearly has a kidney,
whereas the kidney of Tilapia, a freshwater teleost, does
express a CLC-K gene (44). These suggest that gene diversities
for CLC-K and CFTR ion channels that underlie epithelial
transports may have become established during the vertebrate
divergence and adaptations.
In summary, the gene diversities of TRP, CLCN, and amiloride-sensitive channel genes in Ciona are contributed mainly
by multiplications of the genes, apparently in the lineagespecific manner. This diversification in ascidian genes contrasts with the genes of voltage- and transmitter-gated channels, for which the Ciona genome has only a minimum set of
prototype genes with a small number of lineage-specific
duplications.
277
278
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Table 2. Ion channels expressed in early embryogenesis
Channel Name
Gastrula
and
Neurula
Tailbud
Larva
⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹
⫺
⫹
⫺
⫺
⫹
⫺
⫹
⫹⫹⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹⫹
⫹⫹
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫹⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹⫹
⫹
⫺
⫺
⫹
⫹
⫺
⫹
⫺
⫺
⫹
⫹⫹
⫹
⫹
⫹
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫹
⫹
⫹
⫺
⫺
⫹
⫺
⫹⫹
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹⫹⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫺
⫺
⫺
⫹⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹⫹
⫹⫹
⫹⫹⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫹
⫹⫹
⫺
⫹⫹
⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫺
⫺
⫺
⫹⫹
⫹⫹
⫺
⫹⫹⫹
⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫺
⫺
⫺
⫹
⫺
⫺
⫹⫹
⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹
⫺
⫺
⫹
⫺
⫺
⫹⫹
⫹
⫺
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫹⫹⫹
⫺
⫹
⫺
⫺
⫺
⫺
⫹
⫹
⫺
⫹⫹
⫹⫹
⫹
⫹⫹⫹⫹
⫹
⫺
⫹
⫹
⫹
⫹
Ion channel species, their developmental expression profiles during at least
one stage before the tailbud embryo, and the robustness of that gene expression
from strongest (⫹⫹⫹⫹) to weakest (⫹) or absence (⫺), according to
expressed sequence tag (EST) database of the Kyoto University Ciona cDNA
project (58), listed for the genes for which cDNA sequence was available in
cDNA libraries. Ion channel genes expressed only at tailbud and/or larva are
not shown in this list.
genes that are grouped with subfamily members of mammalian
Nav channel genes (40). Two rounds of gene duplication
events are proposed to have occurred in early chordate evolution (22, 52). Much of the gene diversity in voltage-gated ion
channel genes could have arisen by means of these genome
duplications, and as a result might have allowed a greater level
of diversification among ion channel function in ancestral
vertebrates. Further genome comparisons, both between differPhysiol Genomics • VOL
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Ci-TWIK3
Ci-TWIK5
Ci-Kir2/5
Ci-Kvbeta
Ci-KCNQ2/3/4/5
Ci-Eag/HCN/CNG diverged2
Ci-Slo
Ci-BKB1
Ci-CNG1
Ci-CNG2
Ci-Cava2/d1
Ci-Cava2/d2
Ci-Cavbeta
Ci-Nav3
Ci-Cav3
Ci-4domain
Ci-TPC2
Ci-ASIC/degenerin3
Ci-ASIC/degenerin2
Ci-ASIC/degenerin6
Ci-Osm9-related1
Ci-Osm9-related2
Ci-TRPM2/4/8
Ci-TRPC-related4
Ci-TRPC-related2
Ci-TRPA1
Ci-TRPA2
Ci-PKD1
Ci-PKD2
Ci-TRPP-related2
Ci-TRPP-related4
Ci-nAChR-A1
Ci-GluR KaiR-like
Ci-GluR delta1
Ci-RyR
Ci-IP3R
Ci-CLIC
Ci-CLCN1/2
Ci-CLCN6
Ci-CLCN7
Ci-connexin-related1
Ci-connexin-related6
Ci-connexin-related7
Ci-connexin-related9
Ci-connexin-related12
Ci-innexin1
Ci-AQP3
Ci-MIP
Ci-AQP8-1
Egg
Cleaving
Embryo
ent teleosts and between teleosts and basal acraniates, will be
necessary to resolve the details of this diversification with
respect to the Ciona genome, however. In the opposite direction in metazoan evolutionary history, before the split between
protostomes and deuterostomes, action potentials generated by
a sodium conductance mechanism have been reported in
sponges, from a level of metazoan organization lacking neurons (37). Thus electrical excitability and behavior apparently
preceded the evolution of structural nervous systems and were
made possible through the early emergence of voltage-gated
ion channels. The molecular homologies of these channels
found here with those in the basal metazoans are not known,
however, and resolution of this question will again require
further comparative genomic analyses.
The Ciona genome also shows a minimal set of transmittergated channels that are closely related to vertebrate channels.
Even though immunoreactivity of the larval CNS has not been
demonstrated to many neurotransmitters (66), there is biochemical or physiological evidence for the utilization of various neurotransmitters in ascidians, including ACh (51), glutamate (14), and 5-hydroxytryptamine (serotonin; 5HT) (49) as
well as GABA (Ref. 29 and T. Koropatnick and I. A. Meinertzhagen, unpublished observations). Corresponding to this
physiological evidence, our analysis here on gene sequences
suggests that Ciona has genes for receptors to ACh, glutamate,
and 5HT as well as GABAA receptors (Fig. 2). Some speciesdependent variations in the genes for transmitter-gated channels are also seen in Ciona; these include GABAR-␳, GlyR,
5HT3R, muscle-type nAChR, and the gene for a glutamate
receptor of the ␦-type, none of which is present in Drosophila
or C. elegans, but all of which are present in both the Ciona
and human genomes. On the other hand, genes for histaminegated and glutamate-dependent Cl⫺ channels are absent from
both the Ciona and human genomes. The absence of histaminegated channels is in accordance with the absence from the
Ciona genome of histidine decarboxylase, the gene encoding
the synthetic enzyme for histamine (11).
Such differences in the spectrum of transmitter-gated receptor channels are in accordance with both the diversity of
neurotransmitter substances in protostomes and deuterostomes
and the ancient provenance of those substances. In particular,
the finding that the Ciona genome contains genes that putatively code for receptor channels gated by serotonin and
glycine indicates that the establishment of systems utilizing
serotonin and glycine for fast synaptic transmission predated
the origin of urochordates. Comparative pregenomic evidence
has previously been interpreted to indicate that certain amine
and amino acid ligands may already have arisen as signaling
molecules in unicellular forms (3), even though their presence
in forms earlier than true metazoans is, by itself, no evidence
for neurotransmitter action. In this connection, it is noteworthy
that C. elegans GluCl ␤-subunit can also be gated by glycine
(34), suggesting that glycine-sensitive receptors evolved independently in C. elegans and chordates. It seems reasonable to
anticipate that neurotransmitter signaling complexity should
increase in proportion to the requirements imposed by the
number of different classes of neurons in nervous systems of
either extant species or their ancestors. The fact that the
number of transmitter-gated channel genes is fewer in the
genome of Ciona than it is in the genome of Drosophila
possibly reflects, for example, the fact that ascidians have a
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
Ciona Lacks Genes for Fine-Tuning Subunits and for the
Fast Conduction of Excitation
Our survey indicates that, with the exception of BK ␤-, Kv
␤-, and Cav ␤-, ␥-, and ␣2/␦-subunits, most auxiliary subunits
for voltage-gated channels are absent from the Ciona genome.
Auxiliary subunits play critical roles in the fine-tuning of
membrane excitability in recent vertebrates (27), and we therefore assume that such functions are lacking in Ciona. KCNQE
subunits, which are critical subunits for modulating slowly
activating K⫹ channels in cardiac cells and neurons, are also
absent in Ciona. Ciona moreover lacks the gene for Nav
␤-subunits, which induce clustering of Nav channels at the
initial segment and node of Ranvier of myelinated neurons,
consistent with the fact that myelination does not occur in the
ascidian nervous system (30) and that there is no myelinrelated gene in the Ciona genome (11). Thus, either these
features were all lost in ascidians, or, much more likely, the
molecular architecture for fast neuronal conduction must have
arisen after the urochordate branch diverged from the other
chordate line. In support of the latter interpretation, Ciona also
lacks ␣1S-type (or Cav1.1) Ca2⫹ channel and skeletal muscle
Nav channel genes, which are both required for fast twitch in
vertebrate muscles. Because the Fugu genome seems to contain a set of genes for rapid electrical conduction, including
genes of the skeletal muscle type Cav channel and Nav channel
as well as the Nav ␤-subunit gene, we conclude that these
Physiol Genomics • VOL
22 •
genes all arose around the emergence of, and have been
conserved in, vertebrates. These findings also suggest that the
innovation of both Nav ␤-subunit and myelin-related genes
may have arisen concurrently during the course of chordate
evolution, in which these genes were required to establish
vertebrates’ locomotory systems. Examination of amphioxus,
which can dart with extreme rapidity and may therefore also
have fast conduction pathways, but which lacks myelin, may
be illuminating.
Contrast in gene Diversities Between Neuronal Excitability
Channels and Channels Related to Homeostatic Control in
the Ciona Genome
In contrast to the channel genes that are related to neuronal
excitability and synaptic transmission, channels related to homeostasis of electrolyte and fluid volume show a more remarkable pattern of diversity, one that also reveals branches unique
to ascidians. The numbers of TRP-related channels, connexins,
and amiloride-sensitive cation channels in the Ciona genome
are comparable to those recorded in the human genome. Why
then are homeostasis-directed channel genes so diverse in
ascidians relative to neuronal channel genes?
The wide gene diversity of TRP-related channels, connexins,
Cl⫺ channels, and amiloride-sensitive cation channels might
reflect the evolutionary history of adaptations of urochordates
to changes in their physical environment. Ciona generally
inhabits the coastal substratum and thus is frequently exposed
to changes in physical conditions such as salinity, osmotic
pressure, and temperature. Such changes are more serious for
the survival of sessile adults than they would be for the
transient lifestyle of the free-swimming larvae. Thus it is
possible that this variety of channels related to homeostasis
may facilitate adaptation of Ciona adults to the intense pressure
of environmental changes. This must await future studies on
the detailed characterization of functional properties of the
relevant channels and their expression patterns in adult tissues.
In addition, this issue will also be assessed by future studies of
homolog genes from other marine invertebrates, especially
other urochordate organisms, including appendicularians and
nonchordate deuterostomes such as sea urchin, as well as in
amphioxus.
Sequential Acquisition of Ion Channel Genes During
Chordate Evolution
Surveys of the Ciona genome reveal the distribution of ion
channel genes in three groups, those unique to vertebrates,
those unique to chordates, and those shared with protostome
invertebrates. From these distributional profiles, predicted major events in the evolution of ion channels are summarized in
Fig. 4. The first group, those genes unique to vertebrates,
contains genes related to advanced epithelial ion transport and
to pain sensation. We could not find any gene for amiloridesensitive cation channels belonging to the ENaC clade, a gene
within a branch of the CLC-K channel genes, or a CFTR-like
gene, even though other members of the amiloride-sensitive
cation channel gene family, the CLCN channel gene family
and the family of ABC-transporter genes, do exist in the Ciona
genome. These genes absent in the Ciona genome putatively
encode channels that are involved in the robust transport of
sodium and chloride ions and especially play critical roles in
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simpler nervous system than that of Drosophila, in which the
order of 100 classes of neurons occur in the visual system alone
(43). On the other hand, the same reasoning cannot hold for C.
elegans, in which, like Ciona, the number of neurons and
possibly also neuron classes is also small (67), while their
transmitter-gated channel genes are considerably divergent
(Table 1).
The predictable aspect of our findings, the conservation of
transmitter-gated channels between ascidian and vertebrate
genomes, is seen not only in the overall homology of genes but
also in the coding sequences for amino acid residues that are
critical to biophysical function. For example, it is well known
that the Ca2⫹ permeability and high Mg2⫹ sensitivity of
NMDA receptors are essential for neuronal plasticity and thus
required for such functions as learning and neural development
(13). The NMDA receptor has a heterooligomeric structure
comprising two types of subunits (NR1 and NR2). Asparagine
(N) in the M2 domain, the pore-forming region, is the critical
site for the Ca2⫹ permeability and Mg2⫹ sensitivity of the
NMDA receptor. The predicted amino acid sequence of both
ascidian NR-like genes has asparagine in the critical site of the
M2 domain, just like all known vertebrate NMDA receptor
subunits. The ascidian channel protein of the NR1/NR2 complex is therefore most likely to be both Mg2⫹ sensitive and
Ca2⫹ permeable, just like the corresponding vertebrate channels. In Drosophila and C. elegans, one of the NR subunits has
a nonasparagine residue in this critical region. The composition
of ascidian putative NMDA channels is not yet known, and
expression studies on Ciona NMDA receptors will be required
to demonstrate that these too are heteromeric and thus to
examine whether the high Ca2⫹ permeability and Mg2⫹ sensitivity of an NR1/NR2 channel complex could be a chordatespecific design.
279
280
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
vertebrate renal, pulmonary, and alimentary function. Ascidians are ammonotelic animals that lack a kidney or other
related excretory organ; excretion and ion transport depend
instead on other organs such as the branchial sac and esophagus (16). A pulmonary alveolar epithelium is also absent in
ascidians. On the other hand, a prototype gene putatively
encoding an ATP-dependent Kir channel (Ci-Kir2/5), which is
essential for renal function in mammals, is present in the Ciona
genome but is found in neither the Drosophila nor C. elegans
genomes (Table 1, Fig. 4). It is also noteworthy that the Fugu
genome contains a CFTR-like gene but lacks both CLC-K and
ENaC homologs, even though Fugu does have nephrons,
whereas the kidney in freshwater Tilapia does express a
CLC-K gene (44). These findings suggest that these channels
involved in advanced ion transport, such as for renal function
or respiration, were acquired or sometimes discarded during
the process of chordate evolution, possibly associated with loss
of a marine habit or adaptation to life on land.
The Ciona genome also seems to lack genes related to pain
sensation. The neural mechanisms underlying vertebrate pain
sensation depend on the functions of several specific classes of
ion channels that are exclusively expressed in peripheral sensory neurons. These include capsaicin receptors, Nav1.8,
Nav1.9, and ionotropic purinergic (P2X-type) receptors, counterpart genes for which are all absent from the Ciona genome.
Physiol Genomics • VOL
22 •
Searches of the Fugu genome show that this teleost contains
genes putatively encoding both P2X and capsaicin receptors
(data not shown). Ciona also lacks neurotrophins and their
receptors, which are important for the modulation of pain
sensation. On the other hand, the Ciona genome does have
genes putatively encoding tachykinin receptors and ASICrelated cation channels, as well as a substance P-like gene, that
also mediate the signaling of pain sensation in vertebrates (33).
Without functional studies, it is still premature to conclude that
ascidians lack pain-sensing pathways, but the evolutionary
acquisition of pain-signaling pathways in the chordate lineage
is likely to have depended not only on the acquisition of novel
ion channels but also on the persistence of these ancient
membrane or ligand proteins.
For genes of the second group, those unique to chordates (or
deuterostomes), our survey also highlights the many ion channel genes that are not present in the Drosophila and C. elegans
genomes but conserved between Ciona and vertebrate genomes
(Fig. 4). These genes include those putatively encoding GIRK,
5HT3R, GlyR, BK channel ␤-subunit, Kv ␤-subunit, ASIC
channel, CLCA channel, GluR ␦-like channel, connexin, twodomain type Cav channel, and CatSper-like cation channel.
Even though their status may require revision in the light of
future genome sequencing projects from other species, espewww.physiolgenomics.org
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Fig. 4. Evolutionary events in ion channel
diversification. Summary showing the major
events in the appearance and disappearance of
genes during the radiation from the presumed
deuterostome/protostome ancestor, as predicted by profiles of ion channel genes from
the genomes of the human, Drosophila,
Chaenorhabditis elegans, Ciona, and Fugu.
CHANNEL GENE DIVERSITY OF THE BASAL CHORDATE
ACKNOWLEDGMENTS
We thank Dr. A. Hazama for helpful comments on the gene diversity of
aquaporins and Drs. T. Tomiki and H. Takahashi for discussions. We thank M.
Takagi at the National Institutes for Physiological Sciences for technical
support.
Present address of Y. Hara, T. Yoshida, and Y. Mori: Laboratory of
Molecular Biology, Dept. of Synthetic Chemistry and Biological Chemistry,
Graduate School of Engineering, Kyoto Univ., Kyoto, Japan.
Present address of H. Watari: Graduate Program in Neurobiology and
Behavior, Univ. of Washington, Seattle, WA.
Physiol Genomics • VOL
22 •
Present address of M. Nishida: Dept. of Pharmacology and Toxicology,
Graduate School of Pharmaceutical Sciences, Kyushu Univ., Higashiku,
Fukuoka, Japan.
GRANTS
This research was supported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology of Japan
to Y. Okamura and Y. Mori (Grant-in-Aid for Creative Scientific Research;
2001–2006, no. 13GS0016), Y. Okada (no. 14207002), and N. Satoh (no.
12202001) and from the Natural Sciences and Engineering Research Council
of Canada to I. A. Meinertzhagen (no. OPG0000065).
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