TMEM120A/TACAN inhibits mechanically activated PIEZO2 channels
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Evaluation Summary:
This manuscript will be of interest people studying mechanosensation (particularly touch and pain sensation) and ion channels. The authors use a combination of electrophysiology and imaging to provide evidence that the force-gated ion channel Piezo2 is negatively regulation by the enzyme Tmem120A/Tacan. The results from the heterologous expression aspects of study are relatively robust and may have potential impact on the field. The physiological relevance of these findings await further investigation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
PIEZO2 channels mediate rapidly adapting mechanically activated currents in peripheral sensory neurons of the dorsal root ganglia (DRG), and they are indispensable for light touch and proprioception. Relatively little is known about what other proteins regulate PIEZO2 activity in a cellular context. TMEM120A (TACAN) was proposed to act as a high threshold mechanically activated ion channel in nociceptive DRG neurons. Here, we find that Tmem120a coexpression decreased the amplitudes of mechanically activated PIEZO2 currents and increased their threshold of activation. TMEM120A did not inhibit mechanically activated PIEZO1 and TREK1 channels and TMEM120A alone did not result in the appearance of mechanically activated currents above background. Tmem120a and Piezo2 expression in mouse DRG neurons overlapped, and siRNA-mediated knockdown of Tmem120a increased the amplitudes of rapidly adapting mechanically activated currents and decreased their thresholds to mechanical activation. Our data identify TMEM120A as a negative modulator of PIEZO2 channel activity, and do not support TMEM120A being a mechanically activated ion channel.
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Evaluation Summary:
This manuscript will be of interest people studying mechanosensation (particularly touch and pain sensation) and ion channels. The authors use a combination of electrophysiology and imaging to provide evidence that the force-gated ion channel Piezo2 is negatively regulation by the enzyme Tmem120A/Tacan. The results from the heterologous expression aspects of study are relatively robust and may have potential impact on the field. The physiological relevance of these findings await further investigation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
A great deal of effort has gone into identifying and studying the role force-gated ion channels in somatosensation. By far the best understood is molecule is Piezo2 which is required for mechanotransduction in a wide range of mechanoreceptors including most touch neurons and proprioceptors. Notably, the majority of responses to high intensity mechanical stimuli (i.e. mechanical pain) remain in mice with conditional ablation of Piezo2 expression and in human subjects who have inherited loss of function mutations in this gene. Very recently, Tmem120a, renamed TACAN, was proposed to function as a slowly adapting stretch-gated ion channel and be required for normal mechanical pain responses. However, this view has been challenged by a series of preprints. In this current study, Del Rosario and colleagues …
Reviewer #1 (Public Review):
A great deal of effort has gone into identifying and studying the role force-gated ion channels in somatosensation. By far the best understood is molecule is Piezo2 which is required for mechanotransduction in a wide range of mechanoreceptors including most touch neurons and proprioceptors. Notably, the majority of responses to high intensity mechanical stimuli (i.e. mechanical pain) remain in mice with conditional ablation of Piezo2 expression and in human subjects who have inherited loss of function mutations in this gene. Very recently, Tmem120a, renamed TACAN, was proposed to function as a slowly adapting stretch-gated ion channel and be required for normal mechanical pain responses. However, this view has been challenged by a series of preprints. In this current study, Del Rosario and colleagues investigate the function of TACAN and find, similar to the other preprints, that heterologous expression of TACAN does not endow cells with any mechanosensitive currents. However, the authors make the unexpected observation that TACAN expression significantly reduces Piezo2 function but not other mechanosensitive ion channels (Piezo1 and Trek-1). The authors go on to provide TIRF imaging evidence that this effect is not due to Piezo2 trafficking to the plasma membrane or Tacan overexpression results rearrangements of the cytoskeleton. Finally, they show Tacan and Piezo2 are co-expressed in DRG neurons and the siRNA-mediation reduction of Tacan expression in neurons alters the relative proportion of mechano-sensitive neurons characterized based on their in vitro adaption properties.
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Reviewer #2 (Public Review):
In this manuscript, Del Rosario et al. use electrophysiology and imaging to show that TMEM120A, a putative high-threshold mechanosensitive ion channel (also named TACAN), does not confer mechanosensitive currents to heterologous cells. Instead, they find evidence that TACAN negatively regulates the mechanosensitive ion channel Piezo2, but importantly, not other mechanosensitive ion channels (Piezo1 or TREK-1). They also find, contrary to a previous report, that siRNA mediated knockdown of TACAN does not reduce the proportion of slowly adapting mechanosensitive currents in DRG neurons (but see below for related concerns).
Overall, the potential impact of this manuscript is extremely high. Their extensive electrophysiological analysis adds to a growing body of preprints suggesting that TACAN is not, in fact, …
Reviewer #2 (Public Review):
In this manuscript, Del Rosario et al. use electrophysiology and imaging to show that TMEM120A, a putative high-threshold mechanosensitive ion channel (also named TACAN), does not confer mechanosensitive currents to heterologous cells. Instead, they find evidence that TACAN negatively regulates the mechanosensitive ion channel Piezo2, but importantly, not other mechanosensitive ion channels (Piezo1 or TREK-1). They also find, contrary to a previous report, that siRNA mediated knockdown of TACAN does not reduce the proportion of slowly adapting mechanosensitive currents in DRG neurons (but see below for related concerns).
Overall, the potential impact of this manuscript is extremely high. Their extensive electrophysiological analysis adds to a growing body of preprints suggesting that TACAN is not, in fact, an ion channel, and go further to suggest a novel role of this membrane protein in regulating Piezo2. I do have several comments that must be addressed, specifically regarding the use of TIRF to quantify membrane expression and the analysis of the electrophysiological data.
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Reviewer #3 (Public Review):
Del Rosario et al. investigate the function of the protein TACAN (TMEM120A) in cell lines and in sensory neurons from the dorsal root ganglion (DRG). TACAN was recently proposed to encode the slowly adapting (excitatory) mechanosensitive channel in sensory neurons, which is a candidate for detecting noxious mechanical stimuli (Beaulieu-Laroche et al., 2020, cited). Del Rosario et al. show that co-expression of TMEM120A reduced Piezo2 currents but did not inhibit two other well-known mechanosensitive channels, Piezo1 and TREK1. They went on to investigate the effects of TMEM120A siRNAs on endogenous mechanosensitive currents in DRG neurons. They show that siRNA-mediated knockdown of TMEM120A has no effect on slowly adapting mechanically activated currents (as argued by Beaulieu-Laroche et al., 2020) but …
Reviewer #3 (Public Review):
Del Rosario et al. investigate the function of the protein TACAN (TMEM120A) in cell lines and in sensory neurons from the dorsal root ganglion (DRG). TACAN was recently proposed to encode the slowly adapting (excitatory) mechanosensitive channel in sensory neurons, which is a candidate for detecting noxious mechanical stimuli (Beaulieu-Laroche et al., 2020, cited). Del Rosario et al. show that co-expression of TMEM120A reduced Piezo2 currents but did not inhibit two other well-known mechanosensitive channels, Piezo1 and TREK1. They went on to investigate the effects of TMEM120A siRNAs on endogenous mechanosensitive currents in DRG neurons. They show that siRNA-mediated knockdown of TMEM120A has no effect on slowly adapting mechanically activated currents (as argued by Beaulieu-Laroche et al., 2020) but increased the proportion of rapidly adapting currents (as expected from the expression study), supporting the idea that TMEM120A may act as a negative modulator of Piezo2 channel activity. While the authors succeeded in demonstrating the potential modulatory effects of TMEM120A on Piezo2 channel activity, they failed to demonstrate the physiological relevance of this potential modulation in native sensory neurons.
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