Wednesday, June 4, 2014

Sometimes, Cold Hurts

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."

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.
These are the words of Albert Schweitzer, physician, theologian, philanthropist, and organist. Yep – he played a mean organ. Albert was born in the Alsace part of France in 1875, and was raised in a household of ministers and musicians. He turned out to be both - but so much more.

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?

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.
The sensor ion channel that I speak of is the TRPA1. Studies assign it a role in pain sensation – just like TRPV1 for heat. But what stimulates it to generate a pain signal?

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.

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.
As for the “1” in TRPA1, all I can say is that they must be anticipating the discovery of more TRPs of this type, although right now it stands alone. It’s kind of weird though, you don’t call a dad “Sr.” if there is no “Jr.” so why is there a TRPA1 if there are no other TRPA’s?

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.

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.
An earlier study (2012) also showed that activity from TRPV1-4 receptors could modulate the activity of TRPA1. Gentle warm temperatures could desensitize TRPA1 and therefore keep pain from being felt. Could this be one of the ways that warm compresses work against pain?

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.

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.
In the same set up, blocking TRPM8 channels did result in a hypothermia (mouse bodies did not initiate a warming response to cold temperatures). So, the authors concluded that while TRPA1 does cause pain in response to cold, it doesn’t start or participate in a program to warm the mice.

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.

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.
The Peruvian green velvet tarantula (Thrixopelma puriens) has a peptide in its venom that is the first identified peptide (protein) TRPA1 antagonist. What’s it doing in venom? One of the purposes of venom is to cause pain – pain is a great teacher – enough pain and you won’t attack that spider again. But here is a potential pain killer in the venom, maybe the green velvet tarantula is trying to kill its prey, but doesn’t want to cause undue stress to its victim. Is that how evolution works?

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.

de Oliveira, C., Garami, A., Lehto, S., Pakai, E., Tekus, V., Pohoczky, K., Youngblood, B., Wang, W., Kort, M., Kym, P., Pinter, E., Gavva, N., & Romanovsky, A. (2014). Transient Receptor Potential Channel Ankyrin-1 Is Not a Cold Sensor for Autonomic Thermoregulation in Rodents Journal of Neuroscience, 34 (13), 4445-4452 DOI: 10.1523/JNEUROSCI.5387-13.2014

Spahn V, Stein C, & Zöllner C (2014). Modulation of transient receptor vanilloid 1 activity by transient receptor potential ankyrin 1. Molecular pharmacology, 85 (2), 335-44 PMID: 24275229

Benemei S, Fusi C, Trevisan G, & Geppetti P (2014). The TRPA1 channel in migraine mechanism and treatment. British journal of pharmacology, 171 (10), 2552-67 PMID: 24206166

Gui J, Liu B, Cao G, Lipchik AM, Perez M, Dekan Z, Mobli M, Daly NL, Alewood PF, Parker LL, King GF, Zhou Y, Jordt SE, & Nitabach MN (2014). A tarantula-venom peptide antagonizes the TRPA1 nociceptor ion channel by binding to the S1-S4 gating domain. Current biology : CB, 24 (5), 473-83 PMID: 24530065

For more information or classroom activities, see:

Albert Schweitzer –

Protein domains/motifs –

Pervian green velvet tarantula –



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  2. Such a nice blog and I appreciate your all efforts about your thoughts. It’s really good work. TRPV1