Animals, including humans, can sense mechanical force in a myriad of forms―as light touch, vibration, and even muscle tension, among others. Yet all of these seem to be detected by a single ion channel, known as Piezo2.
Now, the labs of Alexander Chesler, National Center for Complementary and Integrative Health, Bethesda, US, and Mark Hoon, National Institute of Dental and Craniofacial Research, Bethesda, US, together show that Piezo2 comes in many varieties, potentially explaining the full range of mechanotransduction. The researchers find that RNA encoding the channel undergoes alternative splicing, a process that allows the mouse Piezo2 gene to yield more than a dozen versions of the protein, with specific variants being expressed by specific cell types. The study also confirmed that the splicing takes place in human tissues.
“Showing that the Piezo2 gene is heavily spliced gives rise to the idea that there might be a lot more diversity in mechanotransduction than we previously thought,” says Jorg Grandl, Duke University, Durham, US, who was not involved in the study.
Cheryl Stucky, Medical College of Wisconsin, Milwaukee, US, agrees, adding that this phenomenon “could underlie why certain mechanoreceptors have such low thresholds, why they are rapidly adapting versus slowly adapting, and why they are vibrational sensors. And, it’s really cool that they did this in human dorsal root ganglia and found similar results. That’s very comforting for the field.” Stucky also did not take part in the research.
The results were published December 5 in Cell Reports.
New variants
In 2014, Ardem Patapoutian, Scripps Research Institute, La Jolla, California, US, and colleagues revealed Piezo2 to be the long sought-after molecule capable of transducing mechanical force into electrical potentials (Ranade et al., 2014). Soon after, Hoon found himself asking, Which sensory neurons express this new mechanosensor?
To find out, co-first author Leah Pogorzala, Hoon, and colleagues began by amplifying messenger RNA (mRNA) encoding Piezo2. In the process, they noticed some strange sequences. Compared to mRNA isolated from the lung or bladder, which matched previously published sequences, transcripts from the trigeminal ganglion (TG) pointed to several variants arising from the alternative splicing of five exons. When the researchers then used single molecule real-time (SMRT) sequencing to detect long stretches of RNA and determine the frequency of each variant, they discovered that the lung and bladder mainly had only one isoform, dubbed V2. In contrast, the TG possessed 17, if not more.
Despite the fact that the three-dimensional structure of Piezo2 has gone unmapped, the researchers still reasoned that the observed splicing likely affected the channel’s intracellular domain. They arrived at this conclusion by taking into account the similarity between Piezo2 and Piezo1—a related mechanosensor—and the predicted structure of the latter. (Three independent groups have since constructed high-resolution models of Piezo1’s architecture: Guo and MacKinnon, 2017; Saotome et al., 2017; Zhao et al., 2018; Chesler and Szczot, 2018.)
But what purpose did the variation in Piezo2 serve? Enter Chesler, who has studied patients with mutations in the Piezo2 gene (see PRF related news story; Chesler et al., 2016). “The first thing that came to mind was, ‘Let’s express these new sequences and see if they even make functional, stretch-gated ion channels. And if they do, do they look like what we believed Piezo2 channels look like?’ ”
Ultimately, co-first author Marcin Szczot, Chesler, and colleagues recorded mechanically activated currents generated by V2 and another variant called V14 in human embryonic kidney cells, since these two variants differed most in the alternatively spliced exons they included. Upon indenting the membrane of the cells, the researchers found that both channels responded, and did so in a way that reflected the strength of the applied force. In other words, they acted as mechanosensors.
But the variants also differed in several respects. V2 showed a higher calcium permeability than V14, and it inactivated at a slower rate. Additionally, V14 alone had a mechanical threshold that could be lowered by intracellular calcium. It seemed, then, that alternative splicing of five exons could adjust the electrophysiological properties of Piezo2.
Solving a riddle
Prior to his collaboration with Hoon, Chesler had been using a newly developed in situ hybridization technique called RNAscope, which can label mRNA as short as 30 base pairs. “We were interested in pushing this technology,” said Chesler.
Fortunately, Hoon’s discovery of the Piezo2 variants provided an ideal opportunity to do so, allowing for visualization of mRNA transcribed from the alternatively spliced exon E35 in certain classes of TG neurons. While this mRNA could not be detected in neurons critical for mechanical pain, it was enriched in neurons involved in touch and proprioception. Sequencing data confirmed this finding, suggesting that even among sensory neurons, specific cell types carry specific variants.
Together, the results could solve one of the riddles of mechanotransduction—how are so many kinds of mechanical force sensed by the same protein? Researchers first encountered this puzzle in mice (e.g., Ranade et al., 2014; Woo et al., 2015). But in 2016, its relevance to humans became clear when Chesler and his colleagues described two patients with loss-of-function mutations in the gene encoding Piezo2. Both had deficits in touch discrimination, vibration sensing, and joint proprioception.
“This molecule is acting as a stretch sensor in different cell types, to underlie different types of sensations,” explained Chesler. “I think the main thing our study indicates is that one of the ways that Piezo2 gets specialized in different cells is at the molecular level.”
This could be as true in humans as it is in mice. Indeed, when the researchers sequenced transcripts from human lung and DRG, a similar story emerged. The predominant variant in the lung resembled V2, and the number of variants in the DRG reached 16.
“You hope for that, but you always worry about how different human results are going to be,” said Stucky. “It seems like mechanotransduction is such a fundamental physiological process that I would think it would be highly conserved between mice and humans, but it sure is good to have the report that it is.”
“I predict there are going to be a lot of future studies that will emerge from this one,” said Grandl.
Piezo2 and pain
Another mystery surrounding Piezo2 concerns its connection to pain. Piezo2 mRNA was present in the TG, including in several populations of nociceptors, but as Chesler and colleagues have previously noted, patients with the protein genetically disabled have normal acute pain sensation, as do Piezo2 knockout mice (Chesler et al., 2016; Ranade et al., 2014).
But now, with the knowledge of how broadly Piezo2 is expressed across tissues in the body, and of its numerous splice variants, Chesler and Hoon are rethinking if and how the mechanosensor feeds into pain.
“The studies of the patients are ongoing,” Chesler told PRF. “It’s possible that there are pain phenotypes in these patients, but we just need to look for them.”
Perhaps, the authors suggest, a change in the expression of certain variants in those with the Piezo2 gene unaltered could even lower pain thresholds, causing allodynia or hyperalgesia.
A switch in sensitivity
This suspicion fits with another new study, led by Stefan Lechner, Heidelberg University, Germany. It finds that nearly half of the nociceptors innervating visceral organs and deep somatic tissues carry the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3). Though these cells normally remain silent with mechanical stimulation, they became responsive when treated with the inflammatory molecule nerve growth factor (NGF). And this switch depended on Piezo2, according to the paper published December 12 in Cell Reports.
“The notion that mechano-insensitive afferents contribute to hyperalgesia has been around for awhile,” said Michael Gold, University of Pittsburgh, US, who was not involved with the work. That a subpopulation of these neurons has a neurochemical signature is “a step forward for the field.”
To identify the channels responsible for the acquired sensitivity, co-first authors Vincenzo Prato, Francisco Taberner, and colleagues assessed mRNA levels of several ion channels previously implicated in sensory neuron mechanosensitivity, after incubating cultured CHRNA3 lumbar DRG neurons with NGF for 24 hours. Surprisingly, out of six potential culprits, not one showed an increase when compared to CHRNA3 neurons cultured without NGF. Nonetheless, both the NGF-treated cells and controls expressed Piezo2 to the same extent as low-threshold mechanoreceptors.
As for whether Piezo2 was to blame, the researchers found three pieces of converging evidence. First, mechanically activated currents in CHRNA3 neurons resembled those conducted by the channel. Second, these currents could be blocked with a toxin that targets Piezo2. Lastly, knocking down the expression of Piezo2 with small-interfering RNA prevented NGF from “unsilencing” the neurons.
To Gold, these data could be explained by a scenario where, “for whatever reason, Piezo2 is not normally activated. But that’s just one possibility. The other possibility is that these neurons were always mechanically sensitive, but that the potential produced by activating the mechanotransducer never made it to action potential initiation. So if you had changes in channels that enabled that transducer to generate an action potential, that would be a mechanism for the emergence of mechanosensitivity.”
“It would be very interesting to see if it’s a Piezo2 splice variant that is uniquely sensitive to NGF, and if that splice variant is upregulated after inflammation,” said Stucky.
Matthew Soleiman is a science writer residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
Image: Szczot et al., 2017
