Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neuron. Author manuscript; available in PMC 2011 Aug 12.
Published in final edited form as:
PMCID: PMC2928144

C. elegans TRP family protein TRP-4 is a pore-forming subunit of a native mechanotransduction channel


Mechanotransduction channels mediate several common sensory modalities such as hearing, touch, and proprioception; however, very little is known about the molecular identities of these channels. Many TRP family channels have been implicated in mechanosensation, but none of them has been demonstrated to form a mechanotransduction channel, raising the question of whether TRP proteins simply play indirect roles in mechanosensation. Using C. elegans as a model, here we have recorded a mechanosensitive conductance in a ciliated mechanosensory neuron in vivo. This conductance develops very rapidly upon mechanical stimulation with its latency and activation time constant reaching the range of micro-seconds, consistent with mechanical gating of the conductance. TRP-4, a TRPN (NOMPC) subfamily channel, is required for this conductance. Importantly, point mutations in the predicted pore region of TRP-4 alter the ion selectivity of the conductance. These results identify TRP-4 as the first TRP protein that functions as an essential pore-forming subunit of a native mechanotransduction channel.


The activity of mechanosensitive channels has been detected in diverse cell types, including both excitable and non-excitable cells (Gillespie and Walker, 2001). These channels can be activated by a variety of mechanical stimuli, ranging from sound to pressure, stretch, and gravity (Christensen and Corey, 2007; Gillespie and Walker, 2001). In the nervous system, several sensory modalities such as hearing, touch and proprioception are believed to be mediated by this type of channels (Christensen and Corey, 2007; Gillespie and Walker, 2001). It has been proposed that these channels are directly gated by force to transduce mechanical stimuli into electrical signals, thereby acting as the transduction channels in mechanosensation (Christensen and Corey, 2007; Gillespie and Walker, 2001).

Despite their prevalence in diverse tissues and organisms, very little is known about the genes encoding mechanotransduction channels in metazoans (Christensen and Corey, 2007). The stretch-sensitive Msc channels found in bacteria appear to be absent in the animal kingdom (Gillespie and Walker, 2001; Kung and Blount, 2004). Thus far, merely two types of proteins have been demonstrated to form mechanotransduction channels in metazoans: the touch-sensitive ENaC family Na+ channel MEC-4/MEC-10 in C. elegans and the stretch-sensitive two-pore-domain K+ channels TREK-1/TRAAK in mammals (Honore et al., 2006; O’Hagan et al., 2005). The molecular identities of the vast majority of mechanotransduction channels, however, remain elusive (Christensen and Corey, 2007; Sharif-Naeini et al., 2008).

TRP (transient receptor potential) channels were first identified in Drosophila and later found to constitute a conserved superfamily of cation channels found in nearly all eukaryotes (Montell, 2005; Montell and Rubin, 1989). TRP family channels have recently emerged as the leading candidates for mechanotransduction channels (Christensen and Corey, 2007; Sharif-Naeini et al., 2008). Among the seven TRP subfamilies (Montell, 2005), nearly every subfamily has members that have been implicated in mechanosensation. These include TRPC (TRPC1, TRPC5 and TRPC6), TRPV (TRPV1, TRPV2, TRPV4, OSM-9, OCR-2, NAN, and IAV), TRPM (TRPM4 and TRPM7), TRPN (TRPN1/TRP-4/NOMPC), TRPA (TRPA1 and Painless), and TRPP (TRPP2/PKD2), as well as the more distantly related Yvc1/TRPY1 (Christensen and Corey, 2007; Gottlieb et al., 2008; Sharif-Naeini et al., 2008). However, a large body of conflicting results has been reported to argue against a direct role of many of these candidates in mechanosensation (Christensen and Corey, 2007; Sharif-Naeini et al., 2008; Yin and Kuebler, 2010). In particular, there is no evidence showing that any of these TRP proteins is mechanically-gated and forms the pore of a transduction channel (Christensen and Corey, 2007). Thus, the question arises as to whether TRP channels merely play an indirect role in mechanosensation; for example, they may act downstream to amplify or modulate the signal from the transduction channel or other components in the pathway (e.g. Gq/PLCβ and PLA2) (Christensen and Corey, 2007; Gottlieb et al., 2008; Mederos y Schnitzler et al., 2008; Sharif-Naeini et al., 2008; Watanabe et al., 2003).

One main challenge lies in the relative difficulty of functionally reconstituting mechanotransduction channels in heterologous systems. Unlike voltage-, ligand-, or temperature-gated channels, the proper function of many mechanotransduction channels probably requires tethering of the channel to the cytoskeleton and/or extracellular matrix, as well as the function of auxiliary subunits, a setting that is difficult to recapitulate in heterolgous systems (Christensen and Corey, 2007; Gillespie and Walker, 2001; Gottlieb et al., 2008).

Here we sought to attack this question in vivo. We resorted to the nematode C. elegans, a popular genetic model organism that encodes 17 TRP family genes covering all of the seven TRP subfamilies and possesses mechanosensory modalities such as touch, proprioception and osmosensation (Kahn-Kirby and Bargmann, 2006; Xiao and Xu, 2009). By combining in vivo patch-clamp recording and molecular genetic manipulation, we identified the TRPN channel TRP-4 as the first TRP family protein that acts as an essential pore-lining subunit of a native mechanosensory transduction channel. Thus, TRP family channels can play a direct role in mechanosensation.


Mechanical stimuli evoke mechanoreceptor currents in CEP

We focused on CEP (cephalic neuron), a mechanosensory dopamine neuron with a sensory cilium located at the nose tip of the worm (Sawin et al., 2000). Notably, the CEP cilium is anchored to the cuticle through a small nubbin (Perkins et al., 1986) (Figure 1A). Deflection of the cuticle by mechanical stimuli such as pressure/touch would lead to deflection of the cilium. Under laboratory conditions, this neuron can sense mechanical pressure imposed by bacterial particles (i.e. food source for worms) during locomotion and foraging (Sawin et al., 2000). Activation of CEP neurons presumably leads to release of dopamine, an inhibitory neurotransmitter for locomotion (Sawin et al., 2000). Consequently, worms reduce their locomotion speed to facilitate feeding, a phenomenon that was first described by Sawin et al as the basal slowing response (Sawin et al., 2000). As previously reported (Sawin et al., 2000), dopamine deficient mutants (e.g. cat-2) lacked this behavioral response (Figure S1A).

Figure 1
In vivo patch-clamp recording of mechanoreceptor currents (MRCs) in the dopamine neuron CEP

As a first step, we developed a protocol to record the activity of CEP in response to mechanical stimuli by whole-cell patch-clamp recording (Figure 1A). We carefully exposed the cell body of CEP to recording pipettes by removing a small piece of cuticle in the head while keeping intact the nose tip where the CEP cilium is housed. A glass probe driven by a piezo actuator was used to deliver mechanical stimuli towards the CEP cilium in a rapid manner with precision (Figure 1A). To facilitate recording, we expressed a fluorescent protein as a transgene in CEP to mark its cilium and cell body.

Mechanical stimuli evoked a rapidly adapting mechanoreceptor current (MRC) in CEP (Figure 1B). Removal of the stimulus also elicited a similar current in CEP, though with a smaller amplitude (Figure 1B). Eliminating Cl from the pipette solution did not affect the reversal potential (Figure S1B), consistent with the view that these currents are carried by cation channels (see below). The presence of both “on” and “off” MRCs is similar to that observed in PLM (posterior lateral microtubule cell), a non-ciliated neuron that detects posterior body touch (O’Hagan et al., 2005). However, unlike the ENaC channel-mediated MRCs in PLM that are Na+-selective (O’Hagan et al., 2005), the MRCs in CEP were non-selective with a reversal potential close to 0 mV (Figure 1C). In addition, CEP MRCs appeared to be insensitive to amiloride (Figure S1C-E). By contrast, we found that as previously reported (O’Hagan et al., 2005), the ENaC-mediated MRCs in PLM were blocked by 200 μM amiloride (Figure S1F-H). Furthermore, the I-V relationship of CEP conductance was nearly linear (Figure 1C), another feature distinct from that observed in PLM (O’Hagan et al., 2005). These results indicate that MRCs in CEP are probably mediated by a distinct class of mechanotransduction channels. As the “on” MRC in CEP is more robust than the “off” MRC, we decided to focus our efforts on the “on” MRC.

The amplitude of MRCs in CEP depends on the strength of mechanical stimuli

The amplitude of MRCs in CEP is stimulus-strength dependent (Figure 2A-B). A displacement of 0.5 μm was sufficient to evoke a MRC in CEP, which saturated around 3 μm (Figure 2A-B). Thus, CEP can sense sub-micrometer range of deflection. This sensitivity may allow CEP to detect mechanical attributes from bacterial particles whose size is usually about 1 μm.

Figure 2
The amplitude of MRCs is stimulus-strength dependent and they do not desensitize to repetitive stimuli with long intervals

MRCs in CEP do not desensitize to repeated stimuli with long intervals

To test whether MRCs undergo desensitization, we applied repetitive stimuli to CEP. Repetitive stimulation of CEP (>20 times) at an interval of 2 sec elicited MRCs with similar amplitude, indicating that under such a condition these currents did not desensitize (Figure 2C-D). Interestingly, CEP did show desensitization to repetitive stimuli with short intervals (Figure 2E-G). Under this condition, the amplitude of MRCs evoked by the second stimulus was greatly reduced compared to that triggered by the first stimulus (Figure 2E). However, such a phenomenon was not observed when we extended the stimulus interval (Figure 2F and 2G). The exact mechanism under this observation is not clear. These results demonstrate that though MRCs in CEP rapidly adapt, they do not appear to desensitize to repeated stimuli with long intervals.

MRCs in CEP show rapid activation kinetics and short latency

One of the hallmarks of mechanically-gated channels is that the kinetics of channel activation should depend on the strength of the stimulus (Christensen and Corey, 2007). Namely, the stronger the stimulus is, the faster the channel opens, as larger forces lower the energy barrier to channel opening more efficiently. We found that the activation kinetics of MRCs in CEP was very rapid with a time constant reaching the range of micro-seconds at saturating stimulus levels (650±90 μs at 4 μm). More importantly, this time constant reduced with increasing stimulus strength (Figure 3A), indicating that the activation kinetics of the transduction channel in CEP depends on the strength of the stimulus.

Figure 3
MRCs in CEP show rapid activation kinetics and short latency

Another key feature of mechanically-gated channels is that the latency of channel activation should be shorter than known second-messenger pathways, usually less than 5 ms (Christensen and Corey, 2007). This makes it unlikely the involvement of second-messengers in channel gating. The latency of MRCs in CEP decreased with increasing stimulus strength, reaching the micro-seconds range at saturating stimulus levels (670±40 μs at 4 μm; Figure 3B-C). The short latency and rapid activation kinetics of MRCs in CEP suggest that the underlying transduction channel is mechanically gated.

Single channel conductance

We then estimated the single channel conductance of the transduction channel through non-stationary noise analysis of MRCs in CEP (Figure 4A-B), as it is technically difficult to record single channel events of this type of channels by excised patch. We found that the single channel current was 1.3±0.1 pA (n=7) at −75 mV. Thus, the single channel conductance (γ) appeared to be 16±2 pS. This analysis also estimated that there was an average of 21±2 functional transduction channels in each recorded CEP neuron.

Figure 4
Non-stationary noise analysis

Adaptation extends the dynamic range of CEP mechanosensitivity

Lastly, we characterized the adaptation of MRCs in CEP. Adaptation allows sensory receptors to adjust their response such that they can maintain their sensitivity to new stimuli in the presence of the existing stimulus (LeMasurier and Gillespie, 2005). It is a critical mechanism for sensory receptors to extend their dynamic range of response (LeMasurier and Gillespie, 2005). This feature is observed in several types of sensory receptors such as photoreceptors and hair cells in vertebrates (LeMasurier and Gillespie, 2005). To test this, we first applied a testing stimulus to CEP to record its initial response (Figure 5A). We then delivered an adapting stimulus followed by another testing stimulus on top of the adapting stimulus to examine the responsiveness of CEP (Figure 5A). Though CEP quickly adapted to the adapting stimulus, it can further respond to additional stimuli in the presence of the adapting stimulus (Figure 5A). As a consequence, the stimulus-response curve shifted to the right (Figure 5B). This feature greatly extends the dynamic range of response of CEP.

Figure 5
Adaptation extends the dynamic range of CEP mechanosensitivity

TRP-4 is required for MRCs in CEP

Having characterized MRCs in CEP, we then asked what channels carry this type of currents. TRP-4, a TRPN subfamily channel, appears to be a promising candidate for a number of reasons. TRP-4 is expressed in CEP and is localized to the cilium of CEP where mechanotransduction presumably occurs (Li et al., 2006; Walker et al., 2000); TRP-4 is also required for the basal slowing behavioral response (Li et al., 2006). In addition, both the zebra fish and fly homologues of TRP-4 have been implicated in mechanosensation (Sidi et al., 2003; Walker et al., 2000). For example, extracellular recordings show that the fly TRP-4 homologue TRPN1/NOMPC is required for one of the two components of trans-epithelial currents induced by bristle displacement in bristle organs (Walker et al., 2000). However, because of some technical constraints, it has not been possible to directly record the mechanosensory neuron of fly bristle organs by patch-clamp. Thus, it remains unclear whether TRPN1/NOMPC forms the pore of a mechanotransduction channel in these organs (Christensen and Corey, 2007; Gillespie and Walker, 2001).

The ability to record MRCs in CEP by patch-clamp offers us a unique opportunity to test whether TRP-4 can form a mechanotransduction channel in vivo. We thus recorded CEP in trp-4 mutant worms and found that mutant worms lacked MRCs (Figure 6A-D). Like other neurons, CEP also expresses voltage-sensitive currents such as K+ currents (Davies et al., 2003), but these currents appeared normal trp-4 mutant worms, indicating that trp-4 mutations do not globally affect ionic currents (Figure S2A-C). Behaviorally, we confirmed that as previously reported (Li et al., 2006), mutant worms lacked the basal slowing response (Figure 6E). Expression of wild-type trp-4 cDNA in CEP under a dopamine neuron-specific promoter rescued MRCs in CEP (Figure 6C-D), as well as the basal slowing behavioral response (Figure S1). As a control, expression of ocr-2 cDNA yielded no rescuing effect on either phenotype (Figure 6D-E), though this cDNA can rescue the osmotic and octanol avoidance defects of ocr-2 mutant worms (Figure S2D-E). In addition, as was the case with wild-type worms, the I-V relationship of MRCs in rescued worms was nearly linear with a reversal potential close to 0 (Figure 6F-G). The response latency and activation kinetics of MRCs in rescued worms (latency: 610±110 μs, activation time constant: 590±40 μs at 4 μm, n=6) were also very similar to those in wild-type worms. These results demonstrate that TRP-4 is required for MRCs in CEP.

Figure 6
TRP-4 is required for MRCs in CEP

However, an essential role of TRP-4 for MRCs in CEP does not provide evidence that TRP-4 forms the transduction channel that carries MRCs in this neuron. Many alternative models exist. For example, TRP-4 may be an essential auxiliary subunit of the transduction channel complex; TRP-4 may also be required for the expression, assembly and/or targeting of the transduction channel. To demonstrate that a protein forms the pore of an ion channel that carries a defined conductance, one of the most commonly used approaches is to show that the ion selectivity of the conductance can be altered by manipulating the residues in the predicted pore region of the candidate protein (Hille, 2001). Thus, we decided to apply this approach to TRP-4. One challenge is that while it is relative easy to alter the ion selectivity of selective channels (e.g. Na+ and K+ channels) by rendering them non-selective, it is generally more difficult to do so with non-selective channels (most TRP channels are non-selective).

Mutations outside of the predicted pore region of TRP-4 do not alter the ion selectivity of the transduction channel

TRP family channels are structurally and evolutionarily related to voltage-gated K+ channels, and contain six putative transmembrane domains with both the N- and C-termini in the cytoplasm and a putative pore region between the fifth (S5) and sixth (S6) transmembrane segments (Nilius and Voets, 2005; Ramsey et al., 2006; Venkatachalam and Montell, 2007). We examined the putative pore region in the linker area between S5 and S6 in TRP-4. By analogy to K+ channels, the pore of TRP-4 would comprise a pore helix followed by a selectivity filter (Figure 7A) (Owsianik et al., 2006). As TRP family channels are cation channels, we focused on the negatively charged residues D and E (Figure 7A). We mutated all of the D and E residues in the predicted pore region (Figure 7A). As a control, we also mutated nearly all of the D and E residues outside of the predicted pore region but within the linker area between S5 and S6 (Figure 7A). All of the constructs harboring point mutations were expressed in CEP as a transgene in the trp-4 mutant background using a dopamine neuron-specific promoter, and the transgenic worms were then tested for MRCs in CEP and also for the basal slowing behavioral response (Figure 7B-G).

Figure 7
TRP-4 is an essential pore-forming subunit of the mechanotransduction channel in CEP

MRCs recorded from both wild-type worms and trp-4 mutant worms rescued by a wild-type trp-4 transgene displayed a nearly linear I-V relationship with a reversal potential around +1.5 mV (Figure 6F-G and and7G).7G). This indicates that the transduction channel is non-selective, exhibiting a slightly higher permeability to Na+ than to Cs+ (PCs+/PNa+ =0.94; Na+ and Cs+ were the primary cations in the bath and pipette solution, respectively). For technical reasons, we were unable to determine the calcium permeability (experimental procedures). Similarly, MRCs recorded from both the DED1711-3AAA and E1716A mutants exhibited a reversal potential indistinguishable from that observed in wild-type (Figure 7D and 7G), demonstrating that these residues are not critical for determining the ion selectivity of the transduction channel (Figure 7A). This result is consistent with the prediction that these residues are not part of the channel pore. E1728K, a point mutation in the predicted pore helix (Figure 7A), also did not affect the reversal potential (figure 7D and 7G), supporting the view that pore helix residues do not have a major effect on ion selectivity.

Mutations in the predicted pore region of TRP-4 alter the biophysical properties of the transduction channel, including its ion selectivity

Interestingly, EPD1739-41QPN and EPD1739-41APA, two mutants that affect residues in the putative selectivity filter (Figure 7A), shifted the reversal potential to the left by 14 mV and 12 mV, respectively (Figure 7E and 7G). Thus, these two point mutations reversed the preference of the transduction channel towards Cs+ and Na+ and rendered it more permeable to Cs+ than to Na+ (PCs+/PNa+=1.7 for EPD1739-41QPN), indicating an important role for the residues E1739 and/or D1741 in regulating the ion selectivity of the transduction channel. In addition, unlike wild-type TRP-4 that displaced a nearly linear I-V relationship, these two mutant channels appeared to be a bit inwardly rectifying (Figure 7E), revealing that these residues also affect channel rectification. As both mutant forms of TRP-4 preserved much of the channel function, they rescued the basal slowing behavioral response (Figure 7C). If E1739 and D1741 are indeed important for controlling ion selectivity, more drastic mutations of these two residues may render TRP-4 non-functional. To test this, we mutated E1739 and D1741 to the positively charged residue K (EPD1739-41KPK). Consistent with a critical role of these residues, the EPD1739-41KPK mutation abrogated the function of TRP-4, since no MRC can be detected in CEP (Figure 7B and 7E, and see below). As expected, this transgene also failed to rescue the basal slowing behavioral response (Figure 7C). These results demonstrate that mutations in the predicted pore region of TRP-4 either abolished the function of or altered the biophysical properties of the transduction channel in CEP, including its ion selectivity.

Encouraged by the above results, we generated two additional mutant forms of TRP-4: EPD1739-41AAA and EPD1739-41GGG, both of which mutated three residues in the putative selectivity filter (Figure 7A). These two mutations shifted the reversal potential of MRCs to the left by 27 mV (EPD1739-41GGG) and 26 mV (EPD1739-41AAA), respectively (Figure 7F-G), a greater shift than that caused by EPD1739-41QPN and EPD1739-42APA. Thus, the permeability of the transduction channel to Cs+ is further increased (PCs+/PNa+=2.9 for EPD1739-41 GGG). We also observed that the I-V relations of these two mutants were inwardly rectifying (Figure 7F), a feature distinct from that observed in wild-type worms. This indicates that these two mutations also altered the rectification of the transduction channel. Taken together, our results showed that TRP-4 was required for MRCs in CEP, and that point mutations in the predicted pore region of TRP-4 either abrogated the activity or altered the biophysical properties, particularly the ion selectivity of MRCs in CEP. Thus, TRP-4 appears to be an essential pore-forming subunit of the mechanosensory transduction channel in CEP.

A pore-dead mutant form of TRP-4 blocks the wild-type transduction channel in vivo

To gather further evidence, we reasoned that if TRP-4 is indeed a pore-forming subunit of the transduction channel, then point mutations disrupting the pore function of TRP-4 should not only render the mutant channel non-functional, but also should block the wild-type channel in a dominant-negative manner. This logic is based on the fact that TRP family proteins function as multimeric channels (Strubing et al., 2001; Xu et al., 1997). To test this, we crossed the transgene expressing the “pore-dead” mutant EPD1739-41KPK into the wild-type background. We found that wild-type worms expressing EPD1739-41KPK showed almost no MRCs in CEP (Figure 7H-I), indicating a nearly complete blockade of MRCs by this mutant form of TRP-4. Remarkably, these “wild-type” worms lacked the basal slowing response and behaved just like trp-4 mutant worms (Figure 7J). These data provide further evidence that TRP-4 is an essential pore-forming subunit of the mechanotransduction channel in CEP.


TRP family channels can play a direct role in mechanosensation

In the current report, to overcome the relative difficulty of characterizing mechanotransduction channels in vitro, we turned to the nematode C. elegans, an in vivo system that allowed us to record MRCs in vivo and to manipulate them with molecular genetic tools. We addressed the highly controversial role of TRP channels in mechanotransduction. We identified the TRPN channel TRP-4 as the first TRP family protein that functions as a pore-lining subunit of a mechanotransduction channel in vivo. Importantly, the short latency and rapid activation kinetics of TRP-4 currents are consistent with the view that this type of channels is gated by mechanical forces. Thus, TRP channels can play a direct role in mechanotransduction.

It remains unclear what contributed to the conflicting results with respect to the role of TRP channels in mechanosensation. One possibility is that when expressed in vitro, TRP channels may interact with endogenous mechanotransduction channels and/or other channel-interacting proteins (Gottlieb et al., 2008). Different heterologous systems may express different sets of mechanotransduction channels and interacting-proteins. One recent report has elegantly clarified the role of the polycystin TRP channel TRPP2/PKD2 in mechanosensation by demonstrating that TRPP2 regulates mechanosensation not by acting as a mechanotransduction channel but rather by modulating the activity of a native mechanotransduction channel of unknown molecular identity (Sharif-Naeini et al., 2009).

C. elegans offers a valuable model to interrogate the role of TRP family channels in mechanosensation

C. elegans represents a nice system to study mechanosensation in vivo, due to its amenability to genetic manipulation and short generation time. In particular, the ability to record MRCs in vivo by whole-cell recording, in conjunction with the availability of facile genetic tools, offers a powerful means to dissect the molecular underpinnings of mechanotransduction at the organismal level. The worm genome encodes 17 TRP family members that cover all of the seven TRP subfamilies (Kahn-Kirby and Bargmann, 2006; Xiao and Xu, 2009). These genes are widely expressed in the nervous system, as well as in some non-excitable cells (Kahn-Kirby and Bargmann, 2006; Xiao and Xu, 2009). In addition to dopamine neurons, a number of other ciliated neurons have also been categorized as mechanosensory neurons in hermaphrodites and males, and they all express TRP family genes (Kahn-Kirby and Bargmann, 2006; Xiao and Xu, 2009). For example, The TRPV channels OSM-9 and OCR-2 and the TRPA channel TRPA-1 have been implicated in mechanosensation (Colbert et al., 1997; Kindt et al., 2007; Tobin et al., 2002). Notably, the mammalian TRPA1 and TRPV channels have also been implicated in mechanosensation, but whether these channels play a direct or indirect role in mechanosensation has become a subject of intense debate (Bautista et al., 2006; Christensen and Corey, 2007; Kwan et al., 2006; Sharif-Naeini et al., 2008). It will be very interesting to address this question in C. elegans.

Like voltage, ligand and temperature-gated ion channels, the exact gating mechanisms for mechanically-gated channels are not clear. Two primary models have been proposed: the membrane stretch model and the tether model in which membrane stretch and protein tether pulls open the transduction channel, respectively (Christensen and Corey, 2007). TRP-4 may be gated by one of these mechanisms. It is also possible that these two mechanisms are not mutually exclusive. Notably, the cilium of CEP is anchored to the overlying cuticle through a small nubbin (Figure 1A). This anchor may stretch the cilium membrane upon mechanical deflection. Alternatively, this anchor may act in a manner analogous to the tip linker found in vertebrate hair cells to pull open TRP-4. Like many ion channels, some auxiliary subunits may work in concert with TRP-4 to facilitate channel gating. One such example is the MEC-4/MEC-10/MEC-2/MEC-6 mechanotransduction channel complex (Bounoutas and Chalfie, 2007). In this complex, MEC-4 and MEC-10 line the channel pore while MEC-2 and MEC-6 facilitate the gating of the MEC-4/MEC10 channel by linking it to the cytoskeleton and extracellular matrix, respectively (Bounoutas and Chalfie, 2007). However, whether TRP-4 also functions in a channel complex remains to be determined.

Concluding remarks

In mammals, most of the cell types that have thus far been examined show some form of mechanosensitivity with diverse properties (Sachs, 1991). However, very little is known about the underlying transduction channels in mammalian cells. In fact, among the four major classes of ion channels (i.e. voltage-, ligand-, mechanically-, and temperature-gated), mechanotransduction channels probably represent the largest group of channels whose molecular identities are largely unknown. There are at least 28 TRP family members in vertebrates (Nilius and Voets, 2005; Ramsey et al., 2006; Venkatachalam and Montell, 2007). Our work raises the possibility that other TRP channels may also function as mechanotransduction channels. As TRP proteins often interact to form heteromeric channels (Strubing et al., 2001; Xu et al., 1997), they can potentially encode a large group of mechanotransduction channels with diverse biophysical properties and distinct biological functions.

Experimental procedure

Behavioral analysis

The basal slowing response was recorded using an automated worm tracking system as previously described (Feng et al., 2006; Li et al., 2006). Briefly, L4 hermaphrodites were picked 16 hours before tracking. Animals were tracked for six minutes on NGM plates spread with a thin layer of freshly-grown OP50 bacteria or with supernatant from OP50 culture. For transgenic lines, only non-mosaic worms were selected for tracking. The last four minutes of tacking data were used to compute locomotion velocity.

Molecular biology

trp-4 cDNA was inserted between the dat-1 promoter and an SL2::YFP fragment. SL2 achieves a role analogous to that played by IRES in mammalian expression vectors. This strategy allows for simultaneous expression of TRP-4 and YFP in CEP from a single construct. All point mutations were introduced by site-directed mutagenesis (Quick change) and verified by sequencing the entire trp-4 coding region. Transgenic lines were generated by directly injecting trp-4 cDNA constructs into trp-4(sy695) mutant worms.


Whole-cell recordings were performed on an Olympus microscope (BX51WI) with an EPC-10 amplifier and the Patchmaster software (HEKA) using a protocol described previously (Brockie et al., 2001; Goodman et al., 1998; Liu et al., 2010; Richmond and Jorgensen, 1999; Ward et al., 2008). Briefly, a glass stimulus probe was driven by a Piezo actuator (PI) mounted on a micromanipulator and triggered by the amplifier. Worms were glued to a sylgard-coated coverglass covered with bath solution, and a small piece of cuticle in the worm head was cut open and pinned down to the coverglass to expose the cell body of CEP. Caution was exercised to keep intact the nose tip where the cilium of CEP is housed. This preparation well preserved the function of CEP; however, it did not permit rapid exchange of bath solution surrounding the cilium where TRP-4 is localized (usually takes a couple of minutes to equilibrate). This limited our ability to test the effect of some “toxic” bath solutions (e.g. Ca2+ only solution and Ca2+-free solution) on reversal potential, as the patch was always lost before solution exchange completed. Thus, we were unable to determine the Ca2+ permeability of TRP-4. Perfusion of Gd3+ and La3+ (100 μM) can block CEP MRCs; however, as we were unable to wash out such an inhibitory effect (slow solution exchange) before losing the seal, it is unclear whether this effect is specific (data not shown).

The CEP neuron was identified by a yellow or red fluorescent protein marker expressed as a transgene driven by the dat-1 promoter (Lints and Emmons, 1999). Recording pipettes were pulled from borosilicate glass. The bath solution contains 145 mM NaCl, 10 mM HEPES, 20 mM glucose, 1 mM MgCl2, 1 mM CaCl2, and 2.5 mM KCl (335 mOsm; pH adjusted to 7.3 with NaOH). The pipette solution contains 145 mM CsCl, 10 mM HEPES, 20 mM glucose, 0.25 mM CaCl2, and 5 mM EGTA, 5 mM ATP, 0.5 mM GTP (325 mOsm; pH adjusted to 7.2 with CsOH). In “Cl-free” solution, Cs-gluconate was used to replace CsCl. ATP and GTP were not essential for MRCs in CEP, but their inclusion prolonged the life of the patch. CsCl solution was used (unless otherwise indicated) to block K+ currents, reducing noises during recording and also enabling I-V analysis of MRCs. Voltages were clamped at −75 mV. Current data were sampled at 20-40 kHz. Series resistance and membrane capacitance were both compensated during recording. Liquid junction potentials were also corrected.

To estimate the relative permeability PCs+/PNa+, we used the following equation derived from the Goldman-Hodgkin-Katz model as previously described (Hille, 2001):


where Erev is the reversal potential of the current.

For non-stationary noise analysis, the variance values were plotted against mean current, and the data were fit with the equation as previously described (Hille, 2001):


where δ2 is the variance, i represents the single channel current, and N indicates the number of channels available for activation. This method works best for those channels that do not undergo desensitization to repeated stimuli. The single channel conductance was then calculated as:


where Vm is the holding potential, and Erev is the reversal potential of the current. Most, if not all, worm neurons are known to be nearly isopotential, thus voltage attenuation is minimal (Goodman et al., 1998).

Supplementary Material



We thank H. Xu and A. Kumar for comments on the manuscript, R. Xiao for sequence alignment and structural modeling. Some strains were obtained from the Caenorhabditis Genetics Center. J.G. and Z.X. received support from the Chinese 111 project (B06018). This work was supported by grants from the NIGMS and the Pew Scholar Program (X.Z.S.X.).


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, Yamoah EN, Basbaum AI, Julius D. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents. Cell. 2006;124:1269–1282. [PubMed]
  • Bounoutas A, Chalfie M. Touch sensitivity in Caenorhabditis elegans. Pflugers Arch. 2007;454:691–702. [PubMed]
  • Brockie PJ, Mellem JE, Hills T, Madsen DM, Maricq AV. The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion. Neuron. 2001;31:617–630. [PubMed]
  • Christensen AP, Corey DP. TRP channels in mechanosensation: direct or indirect activation? Nature reviews. 2007;8:510–521. [PubMed]
  • Colbert HA, Smith TL, Bargmann CI. OSM-9, a novel protein with structural similarity to channels, is required for olfaction, mechanosensation, and olfactory adaptation in Caenorhabditis elegans. J Neurosci. 1997;17:8259–8269. [PubMed]
  • Davies AG, Pierce-Shimomura JT, Kim H, VanHoven MK, Thiele TR, Bonci A, Bargmann CI, McIntire SL. A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell. 2003;115:655–666. [PubMed]
  • Feng Z, Li W, Ward A, Piggott BJ, Larkspur E, Sternberg PW, Xu XZS. A C. elegans model of nicotine-dependent behavior: regulation by TRP family channels. Cell. 2006;127:621–633. [PMC free article] [PubMed]
  • Gillespie PG, Walker RG. Molecular basis of mechanosensory transduction. Nature. 2001;413:194–202. [PubMed]
  • Goodman MB, Hall DH, Avery L, Lockery SR. Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron. 1998;20:763–772. [PubMed]
  • Gottlieb P, Folgering J, Maroto R, Raso A, Wood TG, Kurosky A, Bowman C, Bichet D, Patel A, Sachs F, et al. Revisiting TRPC1 and TRPC6 mechanosensitivity. Pflugers Arch. 2008;455:1097–1103. [PubMed]
  • Hille B. Ion channels of excitable membranes. third edn Sinauer Associates, Inc.; Sunderland: 2001.
  • Honore E, Patel AJ, Chemin J, Suchyna T, Sachs F. Desensitization of mechano-gated K2P channels. Proceedings of the National Academy of Sciences of the United States of America. 2006;103:6859–6864. [PMC free article] [PubMed]
  • Kahn-Kirby AH, Bargmann CI. TRP channels in C. elegans. Annual review of physiology. 2006;68:719–736. [PubMed]
  • Kindt KS, Viswanath V, Macpherson L, Quast K, Hu H, Patapoutian A, Schafer WR. Caenorhabditis elegans TRPA-1 functions in mechanosensation. Nature neuroscience. 2007;10:568–577. [PubMed]
  • Kung C, Blount P. Channels in microbes: so many holes to fill. Molecular microbiology. 2004;53:373–380. [PubMed]
  • Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, Corey DP. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction. Neuron. 2006;50:277–289. [PubMed]
  • LeMasurier M, Gillespie PG. Hair-cell mechanotransduction and cochlear amplification. Neuron. 2005;48:403–415. [PubMed]
  • Li W, Feng Z, Sternberg PW, Xu XZS. A C. elegans stretch receptor neuron revealed by a mechanosensitive TRP channel homologue. Nature. 2006;440:684–687. [PMC free article] [PubMed]
  • Lints R, Emmons SW. Patterning of dopaminergic neurotransmitter identity among Caenorhabditis elegans ray sensory neurons by a TGFbeta family signaling pathway and a Hox gene. Development. 1999;126:5819–5831. [PubMed]
  • Liu J, Ward A, Gao J, Dong Y, Nishio N, Inada H, Kang L, Yu Y, Ma D, Xu T, et al. C. elegans phototransduction requires a G protein–dependent cGMP pathway and a taste receptor homolog. Nature neuroscience. 2010;13:715–722. [PMC free article] [PubMed]
  • Mederos y Schnitzler M, Storch U, Meibers S, Nurwakagari P, Breit A, Essin K, Gollasch M, Gudermann T. Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction. The EMBO journal. 2008;27:3092–3103. [PMC free article] [PubMed]
  • Montell C. The TRP superfamily of cation channels. Sci STKE. 2005;2005:re3. [PubMed]
  • Montell C, Rubin GM. Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron. 1989;2:1313–1323. [PubMed]
  • Nilius B, Voets T. TRP channels: a TR(I)P through a world of multifunctional cation channels. Pflugers Arch. 2005;451:1–10. [PubMed]
  • O’Hagan R, Chalfie M, Goodman MB. The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals. Nature neuroscience. 2005;8:43–50. [PubMed]
  • Owsianik G, Talavera K, Voets T, Nilius B. Permeation and selectivity of TRP channels. Annual review of physiology. 2006;68:685–717. [PubMed]
  • Perkins LA, Hedgecock EM, Thomson JN, Culotti JG. Mutant sensory cilia in the nematode Caenorhabditis elegans. Developmental biology. 1986;117:456–487. [PubMed]
  • Ramsey IS, Delling M, Clapham DE. An introduction to TRP channels. Annual review of physiology. 2006;68:619–647. [PubMed]
  • Richmond JE, Jorgensen EM. One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nature neuroscience. 1999;2:791–797. [PMC free article] [PubMed]
  • Sachs F. Mechanical transduction by membrane ion channels: a mini review. Molecular and cellular biochemistry. 1991;104:57–60. [PubMed]
  • Sawin ER, Ranganathan R, Horvitz HR. C. elegans locomotory rate is modulated by the environment through a dopaminergic pathway and by experience through a serotonergic pathway. Neuron. 2000;26:619–631. [PubMed]
  • Sharif-Naeini R, Dedman A, Folgering JH, Duprat F, Patel A, Nilius B, Honore E. TRP channels and mechanosensory transduction: insights into the arterial myogenic response. Pflugers Arch. 2008;456:529–540. [PubMed]
  • Sharif-Naeini R, Folgering JH, Bichet D, Duprat F, Lauritzen I, Arhatte M, Jodar M, Dedman A, Chatelain FC, Schulte U, et al. Polycystin-1 and −2 dosage regulates pressure sensing. Cell. 2009;139:587–596. [PubMed]
  • Sidi S, Friedrich RW, Nicolson T. NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Science (New York, NY. 2003;301:96–99. [PubMed]
  • Strubing C, Krapivinsky G, Krapivinsky L, Clapham DE. TRPC1 and TRPC5 form a novel cation channel in mammalian brain. Neuron. 2001;29:645–655. [PubMed]
  • Tobin D, Madsen D, Kahn-Kirby A, Peckol E, Moulder G, Barstead R, Maricq A, Bargmann C. Combinatorial expression of TRPV channel proteins defines their sensory functions and subcellular localization in C. elegans neurons. Neuron. 2002;35:307–318. [PubMed]
  • Venkatachalam K, Montell C. TRP channels. Annual review of biochemistry. 2007;76:387–417. [PMC free article] [PubMed]
  • Walker RG, Willingham AT, Zuker CS. A Drosophila mechanosensory transduction channel. Science (New York, NY. 2000;287:2229–2234. [PubMed]
  • Ward A, Liu J, Feng Z, Xu XZ. Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nature Neurosci. 2008;11:916–922. [PMC free article] [PubMed]
  • Watanabe H, Vriens J, Prenen J, Droogmans G, Voets T, Nilius B. Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels. Nature. 2003;424:434–438. [PubMed]
  • Xiao R, Xu XZ. Function and regulation of TRP family channels in C. elegans. Pflugers Arch. 2009;458:851–860. [PMC free article] [PubMed]
  • Xu XZS, Li HS, Guggino WB, Montell C. Coassembly of TRP and TRPL produces a distinct store-operated conductance. Cell. 1997;89:1155–1164. [PubMed]
  • Yin J, Kuebler WM. Mechanotransduction by TRP channels: general concepts and specific role in the vasculature. Cell biochemistry and biophysics. 2010;56:1–18. [PubMed]
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...