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. 2010 Aug 16;5(8):e12177.
doi: 10.1371/journal.pone.0012177.

TRPA1 mediates mechanical currents in the plasma membrane of mouse sensory neurons

Daniel Vilceanu  1 , Cheryl L Stucky
Affiliations

TRPA1 mediates mechanical currents in the plasma membrane of mouse sensory neurons

Daniel Vilceanu et al. PLoS One. .

Abstract

Mechanosensitive channels serve as essential sensors for cells to interact with their environment. The identity of mechanosensitive channels that underlie somatosensory touch transduction is still a mystery. One promising mechanotransduction candidate is the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel. To determine the role of TRPA1 in the generation of mechanically-sensitive currents, we used dorsal root ganglion (DRG) neuron cultures from adult mice and applied rapid focal mechanical stimulation (indentation) to the soma membrane. Small neurons (diameter <27 microm) were studied because TRPA1 is functionally present in these neurons which largely give rise to C-fiber afferents in vivo. Small neurons were classified by isolectin B4 binding. Mechanically-activated inward currents were classified into two subtypes: Slowly Adapting and Transient. First, significantly more IB4 negative neurons (84%) responded to mechanical stimulation than IB4 positive neurons (54%). Second, 89% of Slowly Adapting currents were present in IB4 negative neurons whereas only 11% were found in IB4 positive neurons. Third, Slowly Adapting currents were completely absent in IB4 negative neurons from TRPA1-/- mice. Consistent with this, Slowly Adapting currents were abolished in wild type IB4 negative neurons stimulated in the presence of a TRPA1 antagonist, HC-030031. In addition, the amplitude of Transient mechanically-activated currents in IB4 positive neurons from TRPA1-/- mice was reduced by over 60% compared to TRPA1+/+ controls; however, a similar reduction did not occur in wild-type neurons treated with HC-030031. Transfection of TRPA1 in HEK293 cells did not significantly alter the proportion or magnitude of mechanically-activated currents in HEK293 cells, indicating that TRPA1 alone is not sufficient to confer mechanical sensitivity.These parallel genetic and pharmacological data demonstrate that TRPA1 mediates the Slowly Adapting mechanically-activated currents in small-diameter IB4 negative neurons from adult mice. The TRPA1 protein may also contribute to a complex that mediates Transient mechanically-activated currents in small IB4 positive C fiber type neurons.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Examples of subtypes of mechanically-activated currents evoked in small-diameter DRG neurons from adult mouse.
Three general subtypes of mechanical currents were observed: A. Slowly Adapting, B. Rapidly Adapting and C. Intermediate Adapting. Red trace indicates increasing mechanical stimuli in 2 µm increments. Responses were graded such that as the magnitude of the stimulus increased, the amplitude of the current increased proportionally. Because increasing the stimulus magnitude sometimes induced Rapidly Adapting currents to become Intermediate Adapting, these two current subtypes were combined into one group designated “Transient currents” for further analysis.
Figure 2
Figure 2. Examples of IB4 staining in small DRG neurons.
A. A small-diameter neuron that labeled positively for IB4-FITC after patch clamp recording and maximal focal mechanical stimulation. B. A small-diameter neuron that was negative for IB4-FITC labeling after patch clamp recording and mechanical stimulation. Note that there are several IB4 positive neurons at the bottom right corner of the image.
Figure 3
Figure 3. Differential expression of mechanically-evoked currents in IB4 positive and negative neurons from wild type mice.
A. Percentage of small neurons responding to mechanical stimulation with either Slowly Adapting (SA) currents or Transient currents. More IB4 negative neurons responded to mechanical stimuli than IB4 positive neurons (* p<0.05, Fisher's exact test). SA currents were expressed predominantly by IB4 negative neurons; few were found in IB4 positive neurons. B. The average magnitude of the SA currents in IB4 negative neurons was much larger than the one SA current found in an IB4 positive neuron. In contrast, the magnitude of the Transient currents in IB4 positive and negative neurons was similar. Bars represent mean ± SEM.
Figure 4
Figure 4. Slowly Adapting mechanical currents were gone in TRPA1−/− neurons.
A. The total percentage of neurons expressing mechanically-evoked currents was unchanged in TRPA1−/− neurons compared to TRPA1+/+ neurons. B. Slowly Adapting currents were absent in IB4 negative neurons from TRPA1−/− mice compared to wild type controls (** p<0.01, Fisher's exact test). A few SA currents were found in IB4 positive neurons from both TRPA1−/− and TRPA1+/+ mice. C. The magnitude of the Transient currents decreased by over 60% in IB4 positive neurons from TRPA1−/− mice compared to wild type controls (* p<0.05, t-test). The magnitude of the Transient current in IB4 negative neurons was not significantly different in TRPA1−/− neurons compared to TRPA1+/+ controls. Bars represent mean ± SEM.
Figure 5
Figure 5. Pharmacological inhibition of TRPA1 abolishes the Slowly Adapting mechanical currents in IB4 negative neurons.
A. The total percentage of neurons expressing mechanically-evoked currents was unaltered in neurons treated with the TRPA1 channel inhibitor, HC-030031 (10 µM), compared to those treated with vehicle (DMSO 0.02%). B. Slowly Adapting currents were abolished in IB4 negative neurons pretreated with HC-030031 compared to those treated with vehicle (** p<0.01, Fisher's exact test). C. The magnitude of the Transient mechanical currents in either IB4 positive (right) or IB4 negative (left) neurons was not altered by pretreatment with HC-030031. Bars represent mean ± SEM.D. Example of a neuron with a Slowly Adapting current that is subsequently completely inhibited by HC-030031. E. Example of a neuron with a Slowly Adapting current, where in the presence of HC-030031, a small residual Transient current remains.
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