2/13/2013
“Evolution of sodium channels
predates the origin of
nervous systems in animals”
Authors
Ben Liebeskind
Graduate student
Dept. Ecology and Evolutionary Biology
U. Texas, Austin
Benjamin Liebeskind, David M. Hillis, and Harold H. Zakon
PNAS: 2011 May 31;108(22):9154-9
David Hillis
Dept. Ecology and Evolutionary Biology
U. Texas, Austin
PI
Director of center for Computational Biology
And Informatics
NAS
Tree of life poster
PNAS
Harold Zakon
Professor of Neurobiology
Section of Neurobiology
U. Texas, Austin
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NaV Channel, α subunit
Gymnotiformes
|----------------investigated sequence------------------|
Mormyriformes
S4 voltage sensors
P-loop: outer pore
linker III-IV inactivation
© C. D. Hopkins
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Arnegard et al S1
neurogenic
myogenic
myogenic
Arnegard et al S1
Phenotypic variation in myogenic Electric Organs: Eomyo
EOD pulse duration:
200 µs – 30 ms (varies by species and sex)
Used in communication
EOD is a based upon sodium-channel action potentials.
Prediction 3: Selection should have acted specifically on target regions of Scn4aa with functional
consequence for EOD waveform.
How did animals evolve?
From Single Cells to a Vast Kingdom
From single celled ancestors, millions of descendant
multicellular animals, in the geological period known as
precambrian.
Origin of animals was ~800 MYA.
Oldest fossil: Adam Maloof (Princeton) found sponge like
multicellular fossils in Australia. In 635 MY old oil
depoists, cholesterol from sponges.
Precambian explosion (~600 MYA).
Recent evidence from genomes finds chemical sequences
found in multicellular animals long before
multicellularity.
Examples: Capsaspora owczarzaki (choanoflagellates) a
single cell parasite of snails.
With an international genome sequencing project started
in 2007, much is now known about the early ancestors of
multicellular animals.
Carl Zimmer (2011) From Single Cells, a Vast Kingdom Arose NYTimes March 14, 2011
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Which organisms have nervous systems?
Sponges are multicellular, but lack a
nervous system
Animals (except sponges and placozoans)
Placozoans are
multicellular but
lack nervous
system
Trichoplax adhaerens
Placozoan body plan and reproduction.
Placozoan phylogeny from whole
genome analysis
Bayesian phylogeny of metazoans places Trichoplax as the sister group to cnidarians and
bilaterians. Maximum parsimony applied to the same alignment results in a single tree
with the same topology shown here. Posterior probabilities are reported above each
branch; likelihood bootstrap support values are reported below.
M Srivastava et al. Nature 454, 955-960 (2008) doi:10.1038/nature07191
Sequencing and analysis of the 98 million base pair nuclear genome of the placozoan
Trichoplax adhaerens. Whole-genome phylogenetic analysis suggests that placozoans
belong to a 'eumetazoan' clade that includes cnidarians and bilaterians, with sponges as
the earliest diverging animals.
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Earliest long distance, targeted, and rapid
communication via electrically excitable cells is seen
earliest in cnidarians, and in bilaterian animals.
Action potentials required.
Nav ion channels
Ca++v channels are found in single celled
protozoa (Paramecium) are used for intracellular
signaling.
Where did the authors obtain their data for this
study? i.e. how did they obtain gene sequence
data for all of the species they used?
Hypothesis: Nav channels evolved from Cav++
channels for intercellular signals that did not
interfere with intracellular signaling.
Genome databases used in this study
Animals with nerve nets
Nematostella vectensis, an anemone.
Putnam et al. (2007) Sea anemone
genome reveals ancestral eumetazoan
gener epertoire and genomic
organization. Science. 317, 86-94.
Trichoplax adhaerens – placozoan
Amphimedon queenslandica - Sponge
Mnemiopsis leidyi, a ctenophore
Choanoflagellates: unicellular protist
1.1 Billion Years of Evolution Produces Three (4?)
Superfamilies of Ion Channels
Gene dup + dimer
2 TM KL +pore
6TM+p Kv
2x 6TM+p Kv
Another gene dup + dimer
4 x 6TM+p Cav Nav
Monosiga brevicollis
King, N.; Westbrook, M.J.; Young, S.L.; Kuo, A.; Abedin, M.; Chapman, J.; Fairclough, S.;
Hellsten, U.; Isogai, Y.; Letunic, I.; Others, (2008). "The genome of the choanoflagellate
Monosiga brevicollis and the origin of metazoans". Nature 451 (7180): 783–8.
doi:10.1038/nature06617. PMC 2562698. PMID 18273011.
Harold H. Zakon (2012) Adaptive evolution of voltage-gated sodium
channels: The first 800 million years. PNAS.
doi/10.1073/pnas.1201884109
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Evolution of Voltage-gated channels
Primitive K+ channel from bacteria is composed of 4 identical
subunits, each with three alpha helicies of which two are
transmembrane domains (red and blue on left)
KcsA channel in bacteria, from x-ray crystal structure. Doyle et al, 1998.
By contrast, voltage gated channels are composed of a
tetramere of six TM-alpha helicies
Another K channel has two repeats of an
inward rectifying K+ channel. Two of
these are thought to form a channel with 4
p-loops.
Hypothetical Nav structure with 4 domains. Liebeskind et al (2011)
What are the main functional differences between
voltage activated sodium channels and voltage
activated calcium channels at protein structural level?
Selectivity gate in Cav
E = glutamic acid
D = aspartic acid
Hypothetical Nav structure with 4 domains. Liebeskind et al (2011)
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Selectivity gate
E = glutamic acid
D = aspartic acid
Selectivity gate
E = glutamic acid
D = aspartic acid,
Usually E/E/E/E
or E/E/D/D for Cav
D/E/K/A for Nav
D/E/K/A for Nav
Hypothetical Nav structure with 4 domains. Liebeskind et al (2011)
Hypothetical Nav structure with 4 domains. Liebeskind et al (2011)
Results
Goals of this study
• Find voltage-gated ion channel genes in basal animals +
unicellular ancestors.
• Analyze evolutionary history of the genes for Nav and Cav
• Explore pore motif (for ion selectivity)
• Explore inactivation gate
Results
Results
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Δ Choanoflagellates have Na channels
Structure of the Na channels
Open reading frame of mRNA transcript sequenced for
Monosiga, and partial transcripts from Trichoplax, so
the Nav genes are expressed.
But they don’t have a nervous
system.
…and they aren’t multicellular.
The pore contains D/E/E/A/ motif in both
species.
(Intermediate between Cav and Nav as seen in other
invertebrates too)
Domains: Trichoplax beta is incomplete (only 3
domains)
Monosiga brevicollis
Pore loops
Human Nav 2.1
D/E/K/A
The 7.2-kilobase cDNA sequence, designated hNav2.1, predicts a 1682-amino acid
protein that bears 52%, 49%, and 46% overall identity with sodium channels cloned
from rat brain, skeletal muscle, and heart, respectively. [873]
Until recently, all cloned vertebrate voltage-dependent sodium channels exhibited high
sequence homology to one another and appeared to comprise a single multigene
subfamily. An exception is the human Nav2.1 channel proposed to represent a second
Na+ channel (NaCh) gene subfamily since comparison with previously cloned voltagegated NaChs revealed only 40-45% identity. [872]
D/E/E/A
D/K/E/A/
Positively charged S4 segments are present in hNav2. 1, but there are fewer basic
residues in repeat domains 1, 3, and 4 than in other cloned sodium channels.
D/E/E/A/
Nav2.1 is prominently expressed in both heart and uterus. [873]
mNav2.3 mRNA was most abundant in heart and uterus, and the transcript levels in
heart, brain, and skeletal muscle were differentially regulated during development.
D/E/E/A/
D/E/--/A
Brugada syndrome, first described in 1992, has a high risk of sudden cardiac death.
Nav2.1 was strongly expressed in the hearts of patients with Brugada syndrome
E/E/E/A
http://channelpedia.epfl.ch/ionchannels/129
Pore loops
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Sodium Channel Evolution
CONCLUSIONS
Study confirms the presence of ion channels in choanoflagellate and placozoan that
match the key elements of Na channels in animals.
Have both the pore domain and the inactivation domain similar to Na channels.
Alternative hypothesis is that these channels are closer to other channels, not Nav.
The results of this study suggest that Nav existed before multicellularity, before animals.
The paper also suggests that these Nav channels were derived from Cav channels.
Shows that function is coopted from existing gene rather than waiting for a new gene,
new function.
The placement of sponges/cnidarians and bilaterians is consistent with other studies, the
placement of ctenophores (have a nervous system) outside of placozoans, cnidarians,
and bilaterians suggestst that placozoans lost the nervous system
Loss of Nav in sponge, may refelct loss of function during sedintary life style.
Since 2011 another Na channel phylogeny
traced to common ancestor of fungi and
animals
Liebeskind, B. J., Hillis, D. M. and Zakon, H. H. (2012). Phylogeny unites animal sodium
leak channels with fungal calcium channels in an ancient, voltage-insensitive clade. Mol
Biol Evol 29, 3613-6.
The supergroup Opisthokonta includes fungi and animals. Obviously animals have nerves
and muscles, and fungi don’t. What about Na channels?
Sodium Leak Channels (NaLCN) Sodium, Leak, non-selective are 4 x 6TM domain Na
channels which have no voltage sensitivity. For example, NALCN can cause a cell to
become slightly depolarized so that it fires repetitively.
Previous studies: showed that NALCN diverged from voltaged gated channels before Cav
and Nav diversified. Fungi have Ca channels with some similarity.
Conclusions
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