Biology concepts – nature of science, TRPA1,
thermoregulation, noxious sensor, chemical sensor, mutation, protein domains
"As we acquire more knowledge, things do not become
more comprehensible, but more mysterious."
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Albert
Schweitser was an organist without compare. He toured
the
world playing different organs for concerts. Some of these
organs
were huge, this one has five keyboards and dozens of
different
knobs and buttons. The money from his concerts
went
directly to building his hospital complex in Africa
(bottom).
At its height, there were over 70 different buildings.
All
care was, and is, free.
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Schweitzer became a minister, but toured the world's great churches
giving organ concerts for big bucks. He used the money to put himself through
medical school and establish a hospital in French Equatorial Africa in the
1910’s. He expanded his hospital to over 70 buildings and treated up to 500
patients at a time, always funding his efforts with his organ concert money
and the money he made from writing books.
He was awarded the Nobel Peace Prize in 1952. His quote
above is true, whether he was speaking of the secrets of life in the spiritual
or scientific sense. I personally phrase Schweitzer’s sentiment a little
differently. The more we know, the less
we know we know. Scientific knowledge is meant to do two opposite things –
one, answer questions, and two, create questions. Every decent answer we think
we find should bring to mind many more questions.
This naturally occurs when science works at its best, but
the hardest part is knowing if we have an answer. If we aren’t sure about the
answer, we have to keep looking. Of course we always keep looking, but some answers come to be so well supported
that it is not the answer we question but the details of the answer – like
evolution or fundamental forces.
But if the answers are tentative or not well supported, then
how good are the questions that spring from them? Can our questions only be as
good as our previous answers? This I where we're lucky, because bad questions can lead
to good answers, if we keep our minds open to what we see and don’t find just
we are expecting to find.
How does this apply to our discussion of thermoregulation
and heat/cool sensing? Well, what happens when you find a thermosensor that you can’t
get a good answer as to what it does? Makes it hard to design new questions
doesn’t it?
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The
left image is of Thomas Hunt Morgan, an American researcher
who
set out to disprove Darwin’s theory of evolution using fruit
flies.
He chose them because they were cheap and bred quickly –
a
new generation came along every 10 days. He started
inspecting
them for mutations, and then bred the mutants to
normal
flies. The proportion of mutant offspring was EXACTLY
as
Darwin predicted. On the right are two images of different
mutants,
often these are induced using radioactivity. On the
bottom,
you can see a leg growing where an antenna ought to be.
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TRPA1 was first described in drosophila melongaster (fruit
flies). Historically, fruit flies have made great models because they eat cheap and
reproduce quickly. You can read about the history of their use in genetics in a
great book called The Violinist’s Thumb
by Same Keene.
The scientists would induce mutations in flies
(and their subsequent offspring) by giving them radiation or chemicals. They
wouldn’t have any idea which flies were mutated, or what the mutations were, it
was just a shotgun method. They would then study the flies, looking for
abnormal anatomy, abnormal behavior, or abnormal responses to stimuli. In some cases, the mutations were spontaneous, not caused by radiation or the chemicals, but finding them was done the same way, and the ionizing radiation or mutagenic chemicals just made the mutation rate much higher. When
they found a fly with a change, then they would go to work and identify the
mutation.
One mutation they noticed was that some flies wouldn’t avoid
things that should have been painful. Before they knew what gene was involved, they decided to call it painless. Comparing
it to known genes, they found it was the drosophila homolog to a mammalian TRP
called TRPA1 or ANKTM1. TRPV’s and TRPM8 were already known, and they all have
ankyrin repeats, so I don’t know why TRPA1 got the “A” for ankyrin.
Ankyrin is just one of thousands of known protein domains, meaning short
sequences of amino acids that are known to have specific functions. Ankyrin is
often found to mediate protein folding and protein-protein interactions, even
though its own folding is a little out of the ordinary. Most proteins with
ankyrin domains have about 4-6 repeats of the 33 amino acid sequence, but the
parasite Giardia lamblia has a
protein with 34 repeats.
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This
is a computer generated image based on ankyrin repeat
morphology.
The different repeats are good for building a protein
interaction
domain. The interesting thing is that although
most
ankyrin repeats have this shape, they bind different
protein
structures and induce different functions. Where's
the
specificity? Good question - could win you a Nobel Prize.
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So, flies with broken painless
(TRPA1) genes didn’t respond to pain; therefore, TRPA1 must be a gene that codes for
a protein that confers a pain signal. This meshed well with the information
from mammals showing that TRPA1 stimulated pain signals from some chemicals. But was this
all?
This is where the controversy began and still continues. Some
studies find that TRPA1 is a noxious chemical receptor, some say it's a
noxious cold receptor. Some experiments show it to be both, a receptor for pain
from cold and a receptor for pain from chemicals. And then there are those that
show it to be a heat receptor! More on those studies in a couple of weeks – they’re
cool… I mean hot!
Even within mammals, the results can sometimes be very
different. Old studies suggested that TRPA1 was a noxious cold sensor (below
15˚C) in humans, but newer research (2013) shows that while TRPA1 does sense
cold in rats and mice, it isn’t affected by cold in humans or monkeys. Many of
the older reports suggesting that TRPA1 is a noxious cold sensor were based on
studies in mice, so maybe they do act differently in humans.
The other possibility is that they don’t sense intense cold
directly, but work with other TRPs to respond with pain when cold is sensed. A 2014 study showed that TRPA1 modulates TRPV1 activity. The two are often
co-expressed on the same neurons, and they are also activated by many of the
same chemicals. This study shows sensitization of TRPV1 by activation of TRPA1
– so maybe this is why your hands burn when you go out in to the cold for a
long time.
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Here
is a cartoon comparison of TRPV1 and TRPA1. The parts
that
go through the membrane (transmembrane domains)
look
very similar, but you can see many differences
in
the intracellular tails of each. Remember that TRPV1 and
TRPA1
bind many of the same chemical agonists, but look at
the
difference in the ankyrin repeats and the other domains
of
the tails, as well as the cytoplasmic sides of the TM
domains.
These are where specificity occurs.
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So, if TRPA1 modulates TRPV1 and vice versa for pain
sensation, maybe TRPA1 works with TRPM8 to induce noxious cold pain. There have been papers that suggest TRPM8 does sense cold temperatures below 15˚C and when those cells are lost, mice have no aversion to
painfully cold stimuli. It is probable that TRPA1 works with TRPM8 and TRPV1 to elicit pain to cold temperatures.
Maybe we could get some insights into the cold sensing of
TRPA1 if we could find out if it participates in warming the body when it is
cold. TRPV1 is a heat sensor and initiates cooling programs. TRPM8 sense cool
and starts to warm the body – so what about TRPA1?
Well, it looks like we get no help there at all. Even in the
species that are most likely to have noxious cold sensation via TRPA1 (rats and
mice), the channel doesn’t look to be calling for warming responses. In fact, a 2014 study suggests that when TRPA1 ion channels are knocked out in mice, cold temperatures
induced physiologic changes just as if the TRPA1 was there – TRPA1 was not a
participant in inducing warming activities.
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This
cartoon gives you an idea of how TRPA1 can work with
other
TRPs in order to increase pain or bring pain when other
signals
alone might not. This example is with injury and
inflammation.
Some things trigger TRPV1 but the activation
of
TRPV1 can influence TRPA1 activity. This would sensitize
for
more pain. Perhaps this is how pain is generated from
intense
cold, even if TRPA1 doesn’t respond to cold on its
own.
TRPM8 does respond to cold, so maybe it or TRPV1
are
the triggers needed for TRPA1 to bring pain from cold.
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Oh well, like we said at the beginning of the post, the more
we know, the less we seem to know for sure. I think TRPA1 is probably involved
in cold pain, even if it doesn’t sense it directly. But I can’t wait to see
what they find out next.
What we do know is that TRPA1 is intimately involved with
pain. Migraine headaches probably have a TRPA1 component. A 2013 paper summarized the evidence by saying that many migraine triggers are now known to be TRPA1
activators. Many of the endogenous stress activators of TRPA1, like oxidative
damage, electrophilic stress, etc. also act to induce pain. Finally, many of the
drugs and analgesics that work on migraines are being identified as TRPA1
antagonists.
Since it’s evident that TRPA1 doesn’t work in
thermoregulation (see above), maybe we can use antagonists of TRPA1 as pain
drugs without worrying about the hyperthermias and hypothermias associated with
TRPV1 antagonists and TRPM8 antagonists. And wouldn’t you know it, a new antagonist for
TRPA1 has just been discovered in a weird place.
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Meet
the Peruvian green velvet tarantula. It does have a
green
hue on its legs and it is soft and velvety. But it isn’t
from
Peru. It actually lives in northern Chile, south of the
Peruvian
border. Its venom contains a TRPA1 antagonist,
but
the problem is that even though it is not likely to bite
the
hand that feeds it, it will fling urticating hairs at the
drop
of a hat. This is important, as we discussed here.
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One last tidbit about TRPA1 – it could save your life in the
middle of the night. If you block mouse nasal activity of TRPA1, they won’t
wake up in response to formalin, acrolein, or other noxious stimuli that should generate an avoidance response. Would
a house fire be something you need to wake up from – you bet. Another recent study found that TRPA1 sensors in upper airway cells are important for sensing
smoke from wood fires. Don’t hate the TRPA1 because it gives you pain – enjoy
the pain – it’s keeping you safe.
Next week – prepare to throw TRPA1 a party; it’s saving your life in many more ways.
For
more information or classroom activities, see:
Albert
Schweitzer –
Protein
domains/motifs –
Pervian
green velvet tarantula –
Such a nice blog and I appreciate your all efforts about your thoughts. It’s really good work. TRPV1
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