Inside JEB
ii
The mutated amino acid located in the hinge
region had dramatically affected the
enzyme's function, and is one of a raft of
adaptations giving the impostor, Mytilus
galloprovincialis, the upper hand over
Mytilus trossulus in hot water.
10.1242/jeb.02107
Fields, P. A., Rudomin, E. L. and Somero, G.
N. (2006). Temperature sensitivities of cytosolic
malate dehydrogenases from native and invasive
species of marine mussels (genus Mytilus):
sequence-function linkages and correlations with
biogeographic distribution. J. Exp. Biol. 209,
656-667.
whether the ion channel plays a role in the
tunicate's simple heart (p.731).
Searching through the C. intestinalis genome,
Salvador-Recatal, Warren Gallin and
Spencer identified a single Kv4 gene; the
vertebrate channel must have become
duplicated after tunicates diverged from our
family tree. Salvador-Recatal also knew that
an accessory protein, KChIP, modulates the
function of modern Kv4 channels, and
wondered whether our tunicate predecessors
still carry this gene? Trawling through the
genome again, the team was delighted to
discover and clone the first non-vertebrate
KChIP subunit gene.
Curious to know how the tunicate's
potassium channel functions, Salvador-
Recatal, Peter Ruben and Jennifer
Abbruzzese began investigating the ion
channel's electrical properties by injecting
Xenopus egg cells with different
combinations of Kv4 and KChIP RNA.
Knowing that the cells would use the RNA
to produce the ion channel and modulating
protein, the team monitored the electrical
properties of the modified cells and found
that when they introduced the channel
alone, the egg cells generated a significant
potassium current. However, when the team
generated both Kv4 and KChIP proteins in
the cells, they saw a dramatic shift in the
Kv4 channel's electrical properties; KChIP
increased the strength of the egg cell's
potassium current as well as prolonging the
current's duration, suggesting that the
ancient channel contributes to tunicate
heart function. Spencer suspects that
KChIP probably aids insertion of the ion
channel in the cell's membrane, increasing
the number of ion channels in the
membrane, and the potassium current in
turn.
Probing the ion channel's function further,
the team removed the first 32 amino acids
of the Kv4 protein, which interact with
KChIP in vertebrates, to see whether this
affected the tunicate channel's function;
KChIP no longer modulated the truncated
ion channel's function, suggesting that
KChIP interacts with tunicate Kv4 through
the channel's N terminus, just like modern
KChIP. So tunicates invented the blueprint
for Kv4 ion channel modulation while
vertebrates have refined ion channel
function further by expanding their
repertoire of Kv4 genes.
10.1242/jeb.02109
Salvador-Recatal, V., Gallin, W. J.,
Abbruzzese, J., Ruben, P. C. and Spencer, A.
N. (2006). A potassium channel (Kv4) cloned
from the heart of the tunicate Ciona intestinalis
and its modulation by a KChIP subunit. J. Exp.
Biol. 209, 731-747.
that galloprovincialis had left the Pacific for
the warmer Mediterranean a mere 3.5 million
years ago, but what adaptations had the
mussel made during its brief European
sojourn that allowed it to thrive and drive
trossulus out? Peter Fields, returning to
Somero's lab for a brief sabbatical period,
decided to take up the challenge by
investigating one of the mussel's key
metabolic enzymes: cytosolic malate
dehydrogenase (p.656).
According to Fields, malate dehydrogenase is
a well-characterised, and essential,
component of several metabolic pathways,
but the enzyme is also temperature sensitive,
making it an ideal candidate for adaptation to
warmer conditions. However, sequencing the
gene from mussels proved trickier than Fields
had hoped. Little is known about mollusc
genomes, so designing the DNA primers
essential for the sequencing process proved
challenging. Fortunately Somero's colleague,
Andy Gracey, was on hand to guide Fields
through the complex sequence databases
needed to design the primers, before Fields
ran the sequencing reactions on both blue
mussels and another California local, Mytilus
californianus, that lives in the mild waters
where galloprovincialis's and trossulus's
territories overlap.
Having sequenced the three genes, Fields
realised that one position in the gene lit up as
a mutation hot spot; all three species had
completely different amino acids at position
114, the hinge of a loop region essential for
the enzyme's catalytic activity. Fields was
astonished. He explains that he'd investigated
related enzymes in cold adapted species and
never seen mutations at this location before.
What effect would this mutation have on the
enzyme's function?
Instead of extracting the enzyme directly
from each of the three mussels, Fields'
student, Emily Rudomin, expressed all three
proteins in bacteria, allowing her to produce
larger quantities of each enzyme than she
could extract from the mussels. Measuring
each enzyme's kinetics, Fields realised that
each of the enzymes were well adapted to
the water temperature where the mussels
lived. He explains that enzymes that
function well in warm conditions have low
substrate turnover rates and bind their
substrate more tightly than enzymes adapted
to cold conditions, which process their
substrate faster and bind substrate more
loosely. Sure enough, the warm adapted
galloprovincialis' malate dehydrogenase
behaved like a warm adapted enzyme, while
the cold adapted trossulus enzyme
functioned best at low temperatures. Mytilus
californianus, found on the beach outside
Somero's Pacific Grove lab, was optimised
for a mild, intermediate temperature.
TUNICATES SET TREND
FOR POTASSIUM
CHANNEL
Ion channels are proteins that form
miniscule pores in nerve cell membranes,
and when open they generate currents across
the membrane to propagate nerve signals.
Andrew Spencer from the University of
Alberta, Canada, explains that one class of
ion channels, the potassium channels, play a
major role in shaping the action potentials
in many tissues, including heart, and as a
consequence have been subject to strong
natural selection resulting in a wide variety
of potassium channels, each finely tuned to
a particular function. One family of
potassium ion channels found in vertebrate
hearts, the Kv4 voltage-gated potassium
channels, comprises three individual
members (Kv4.1, Kv4.2 and Kv4.3) that
evolved from one of our ancestor's early
Kv4 channels. However, the hearts of
simpler invertebrates only carry a single
Kv4 channel, like our ancestors. Knowing
that sea squirts (tunicates) are our most
ancient living relatives, Vicenta Salvador-
Recatal and Andrew Spencer wondered
whether our tunicate ancestors are more like
invertebrates, with only one Kv4 channel, or
whether the Kv4 gene had been duplicated
early enough in evolutionary history for
tunicates to carry multiple Kv4 ion
channels. Salvador-Recatal decided to
investigate how many copies of the gene the
tunicate Ciona interstinalis possesses, and
THE JOURNAL OF EXPERIMENTAL BIOLOGY