First step toward the holy grail of pain research? Molecular identity of a mechanically gated ion channel, Coste et al., 2010 Science

ResearchBlogging.orgFor as long as I have been in pain research (and long before I ever even thought about pain research) the topic of mechanically-gated ion channels has been a huge deal. The reasons are simple:
1) We obtain information about our environment through touch (among other things) but in certain conditions touch can become painful. We call this allodynia. The problem is we don’t know how this happens (but we have some good ideas — more on this later) and, ostensibly, identifying ion channels involved in mechanosensation would go a long way toward helping us understand this.
2) Certain types of mechanical inputs are painful (e.g. pinch or pin-prick) and these types of input become even more painful after injury. We call this hyperalgesia and, again, we have some good ideas of the processes that underlie this hyperalgesia but, ultimately, without knowledge of the initial transducer of mechanical inputs, it is hard to understand this fully.
3) Time for the most obvious reason, we just flat out don’t know how we feel mechanical stimulation. There are hundreds (or thousands, depending how you think about it) of papers out there on this but, to date, no one has clearly identified a mechanically-gated channel expressed by vertebrate sensory neurons.

Until now. There were all types of rumors flying around about this at IASP. This is not unusual, however, as I have heard similar rumors at SfN and IASP meetings in the past. On Sept 2, a paper was published in Science from the Patapoutian lab at Scripps, La Jolla, that may open the flood gates in terms of describing how mechanical stimulation is transduced into signals that can be transmitted to the CNS.

How’d they do it?
I’m no channelologist (calling Nat) but I know enough to get the basics across. First they devised a method for stimulating and recording mechanically activated (MA) currents in cells. Then they set out to find cells that showed a clear mechanically gated current. They ultimately landed on using Neuro2A cells because they expressed a consistent MA current. They then did microarray studies in all of the cells they tested and used what looks like a subtractive method to determine which genes to examine more carefully. They enriched for genes expressed in their Neuro2A cells but not in cells without a consistent MA current. Then they narrowed these down to ones that had some channel properties (at least 2 membrane spanning motifs) and were either known cation channels or genes of unknown function. This gave them 72 candidates. They then starting knocking down expression of these gene, presumably one by one, in their Neuro2A cells. They found that knockdown of gene Fam38A led to a specific decrease in MA currents in their cells. Because this gene encodes a channel activated by pressure they called it Piezo1 (from the Greek word for pressure).

With Piezo1 in hand they went on to find another member of this family of ion channels which they called Piezo2. They used an informatic approach to learn more about this novel gene family:

Many animal, plant, and other
eukaryotic species contain a single Piezo (Fig. 3A).
Vertebrates have two members, Piezo1 (Fam38A) and Piezo2
(Fam38B). However, the early chordate Ciona has a single
member. Multiple Piezos are also present in the Ciliophora
kingdom: Tetrahymena thermophila has three members;
Paramecium Tetraurelia, six. No clear homologs were
identified in yeast or bacteria. The secondary structure and
overall length of Piezo proteins are moderately conserved,
and similarity to other proteins is minimal. As assayed by the
Transmembrane Hidden Markov Model prediction program
(TMHMM2), all have between 24 and 36 predicted
transmembrane domains (with variability perhaps due to
inaccurate cDNA or transmembrane prediction). The
predicted proteins contain 2100 to 4700 amino acids, and the
transmembrane domains are located throughout the putative
protein (fig. S3).

They also did Northern blots to look at the expression patterns for these channels:

Piezo1 expression was observed in bladder,
colon, kidney, lung and skin (Fig. 3B). This pattern agrees
with northern blot expression analysis in rat (14). Bladder,
colon, and lung undergo mechanotransduction related to
visceral pain (17), and primary cilia in the kidney sense
urinary flux (18). The relatively low amount of mRNA in
DRG suggests that Piezo1 may not account for MA currents
observed there (8, 9, 19–22), but Piezo1 was observed in the
skin, another putative site of somatosensation. Piezo2
expression was observed in bladder, colon and lung as well,
but less abundant in kidney or skin. Strong expression of
Piezo2 was observed in DRG sensory neurons, suggesting a
potential role in somatosensory mechanotransduction.

With this expression data they then set out to look at whether Piezo2 contributes to MA currents in dorsal root ganglion (DRG) neurons. They found that Piezo2 is expressed (at the mRNA level) in a range of DRG neurons that are thought to be involved in mechanosensation. Moreover, knockdown of Piezo2 in these neurons led to a specific ablation of rapidly adapting MA currents in almost all neurons. This suggests that Piezo2 may well play a key role in rapidly adapting MA currents in DRG neurons, at least in vitro. In the author’s own words:

We described a role of Piezo2 in rapidly-adapting
mechanically-activated currents in somatosensory neurons.
Thus Piezo2 has potential roles in touch and pain sensation. Piezo 1 and 2 are expressed in various tissues, and
their homologs are present throughout animals, plants, and
protozoa, raising the possibility that Piezo proteins have a
broad role in mechanotransduction.

Clearly this is not the end of the story for MA channels in DRG neurons (or other tissues) as there are some big holes left unfilled by this otherwise fascinating story. The biggest one, at least in terms of DRG neurons, is that while rapidly adapting MA currents appear to be mediated by Piezo2, there are clearly other types of MA currents present in these neurons that are completely unaffected by knockdown of Piezo2. Moreover, while the in vitro data is highly convincing, there are no indication in the present paper that these channels mediate MA currents in vivo. In the absence of specific inhibitors or a knockout mouse, such studies would be difficult to do, obviously. On the other hand, the identification of these channels opens the door, for the first time, to the possibility of doing these studies in the first place. Its going to be fascinating to follow this area of work (and possibly jump into it) in the coming years.

Finally, why is this such a big deal? I’m sure there are some perfectly good reasons that I will miss but here are a few:
1) The most obvious: this field LOVES channels and also LOVES channels as targets for drug discovery. Now we’ve got some new channels to study and new drugs to make. It’ll be very exciting to see if there are more members of this family of channels and to see the molecules that fall out of drug discovery efforts that target these channels. They’ll likely be fantastic tools and may have some important therapeutic implications.
2) Allodynia: With the identification of these channels maybe we’ll finally get a better handle on allodynia. If you’re wondering what allodynia is, I’ve written about it before, including some explanation of its mechanisms. The prevailing thought in the field is that allodynia is due to changes in the central nervous system that alter the so-called “pain gate” in the dorsal horn. While evidence for this is strong, there are numerous studies that muddle this picture significantly. Pharmacological blockade of newly identified mechanically-gated channels (or knockdown and/or knockouts) could go a long way toward helping us understand the differential contributions of peripheral vs. central mechanisms to allodynia. I am very excited about the prospects for major discoveries in this area from these new findings.
3) Labelled lines for sensory information: The question here is whether sensory information is relayed to the CNS along so-called “labelled lines” or whether individual neurons are able to encode sensory input through as yet to be clearly identified mechanism. This debate has raged for decades. A number of papers have been published recently demonstrating that there is strong evidence for labelled lines, at least for thermal and mechanical information in the pain pathway. However, this work is limited by lack of information on the molecular identity of mechanically-gated ion channels. The present work may open new frontiers in this line of work and afford opportunities to test whether these “labelled lines” change after nerve injury or inflammation.

Coste, B., Mathur, J., Schmidt, M., Earley, T., Ranade, S., Petrus, M., Dubin, A., & Patapoutian, A. (2010). Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels Science DOI: 10.1126/science.1193270

9 responses to “First step toward the holy grail of pain research? Molecular identity of a mechanically gated ion channel, Coste et al., 2010 Science

  1. Pingback: Tweets that mention First step toward the holy grail of pain research? Molecular identity of a mechanically gated ion channel, Coste et al., 2010 Science | JUNIORPROF --

  2. It’s an interesting paper, for sure. I especially appreciate getting the pain/pharmacology perspective. Thanks for that!

    I have to take issue with one part though: “no one has clearly identified a mechanically-gated channel expressed by sensory neurons”. The first one that comes to mind is Marty Chalfie’s work in C. elegans on MEC-4. Another recent case is TRP-4 in C. elegans ( Both act in sensory neurons.

  3. Andre,
    Yes, you are right, and I should have said vertebrate sensory neurons. I’ll correct that.

  4. *wakes up from cold medicine induced haze* Hunh?

    First a disclaimer – our lab and Ardem’s are certainly competitors in many things, and my one of my own papers basically took aim directly at one of his.

    I’ve checked out the currents, and they look very believable. When thinking about recordings of mechanically activated channels, there’s always the worry that when you apply pressure (via either suction through the patch pipette or pushing on the cell) you are simply making the cell membrane transiently leaky. But the way the current adaptation behaves at different potentials suggest this isn’t likely (I especially like the slow decay of the current at positive potentials when the pressure has been removed -e.g. Fig 4B). The responses in varying ionic conditions also support this.

    Sure, as an ion channel geek I think they ought to have shown the ion substitution expts in the main text, as well as the application and removal of the blockers they used. But neither of those impact the conclusion that these Piezo proteins are intimately involved in the mechanically activated current.

    What’s more ambiguous, which they acknowledge, is whether Piezo1/2 actually form the ion conducting pathway, or whether they regulate a separate protein (a la the SUR and Kir6.2). Another somewhat similar example is the story of pICln.

    Demonstrating that Piezos directly mediate the ion conduction will require identification of residues that form the pore. And in some sense it’s a separate point. Even if Piezos are regulators of a separate channel, they are the best instance of a vertebrate protein that’s likely involved in mechanical transduction. Now that there’s a little foothold, there are other techniques that can be used to fish out other important players (any of which might be useful targets for therapeutics).

    • Thanks for the patch explanations Nat…. I was hoping I would lure you in with this one! I agree, this “little foothold” should lead to all kinds of interesting stuff.

  5. Nice summary, but I must give you a bit of a hard time for using one of Carl Zimmer’s banned words in science writing. ;)

  6. that’s a stupid fucking list

  7. Zen, in my defense: A) I’m not a science writer, B) mechanically gated channels have been known as the holy grail in pain research since TRPV1 cloning in 1990’s and C) Its a silly list

  8. Pingback: Pain sensation and sodium channels | medicalneurophys

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