Mechanotransduction in sensory neurons has come under intense investigation in recent years and is forefront in the field of sensory neurobiology 
. Sensory neurons express a wide variety of TRP channels that are thought to be in some manner, sensitive to mechanical force, including TRPA1, TRPV2, TRPV4, TRPC1, TRPC6 and TRPP2 
. Among these, the TRPA1 channel is unique in that it has 18 ankyrin repeats in the N terminus that have been hypothesized to act as a spring when under mechanical stress 
. Here, we investigated the role of TRPA1 in generation of mechanically-activated currents in the plasma membrane of isolated DRG neurons from adult mice using a dual approach: genetic ablation of TRPA1 and acute pharmacological inhibition of TRPA1.
The site of mechanotransduction in sensory neurons is at their peripheral terminals in the end organs (e.g. skin, muscles, viscera). However, the membrane of peripheral nerve endings is not directly accessible to functional investigation. In lieu of this, one widely used approach is to investigate the soma of isolated DRG neurons with the assumption that the soma plasma membrane mimics the peripheral terminal plasma membrane regarding the expression of the same ion channels within the same intracellular milieu. Here we applied focal mechanical stimulation to the soma of isolated DRG neurons from adult mice and reproduced the mechanically-activated currents described in the literature by other groups 
. Further, we classified small-diameter DRG neurons by IB4 staining and found that IB4 negative neurons were significantly more responsive to mechanical stimulation than IB4 positive neurons. Moreover, when we stratified the mechanosensitive currents into Slowly Adapting (SA) currents and Transient currents, we found that most (89%) of the SA currents are found in IB4 negative neurons and few (only 11%) appear in IB4 positive neurons. One plausible explanation is that there is a different expression of ionic protein channels in these two subpopulations of C fiber type sensory neurons (see below). However, an intriguing alternative is that IB4 negative and positive neurons may have different plasma membrane lipid composition and thus, punctuate force or stretch stimuli may distribute differently to the ion channel proteins in the membrane that transduce external mechanical energy into membrane currents. Anecdotally, in our experience, IB4 negative neurons form tight seals in patch clamp recordings significantly faster than IB4 positive neurons (prior to staining), suggesting that IB4 negative neurons inherently possess a more dynamic plasma membrane (Vilceanu and Stucky, unpublished observations).
Mice deficient in TRPA1 have been shown to have deficits in sensing intense force applied to the hindpaw skin in behavioral assays 
. In skin nerve preparations from these TRPA1-deficient mice, C fiber nociceptors fire 50% fewer action potentials to mechanical stimuli applied to skin receptive fields than wild type littermates 
. Local application of a TRPA1 antagonist to skin receptive fields in wild type mice or rats induces similar decreased action potential firing 
. Importantly, TRPA1 is expressed not only by sensory neurons but also in keratinocytes in the epidermis 
. Since behavioral assays and skin nerve experiments both involve application of mechanical stimuli to keratinocytes as well as sensory nerve terminals, the site where TRPA1 physiologically contributes to mechanical activation of sensory neurons in situ
is not clear. TRPA1 may contribute to mechanotransduction via its expression directly in the plasma membrane of the sensory terminal, or by its expression in keratinocytes that closely associate with the sensory terminal endings. Via either location, TRPA1 may ultimately contribute to mechanically-evoked action potentials in the sensory neuron, by directly contributing to transduction of the mechanical stimulus, by modulating the mechanically-activated currents, or by conveying the mechanically-evoked action potentials toward the spinal cord.
Our study indicates that TRPA1 in the sensory neuron plasma membrane participates in the generation of Slowly Adapting mechanically-activated currents. In wild type DRG neurons, SA currents are present predominantly in IB4 negative small-diameter neurons. IB4 negative neurons from TRPA1-deficient mice lack all SA currents. Similarly, pretreatment with a TRPA1 antagonist, HC-030031, inhibited all SA currents in wild type IB4 negative neurons. In some neurons with SA currents that were tested both before and after treatment with HC-030031, a very small residual Transient current remained in the presence of the inhibitor. This finding together with our finding that the total number of mechanically-sensitive neurons does not significantly decrease in the TRPA1−/− strain or in neurons pretreated with HC-030031, suggests that the SA currents may mask a Transient current that is still present in absence of TRPA1.
Our findings are consistent with evidence that SA currents are mediated by non-specific cationic channels in that TRPA1 is known to be a non-specific cationic channel 
. Interestingly however, a few SA currents were still present in IB4 positive neurons from TRPA1-deficient mice and in wild type neurons treated with the TRPA1 inhibitor. These remaining SA currents in IB4 positive neurons must be mediated by mechanically-sensitive channels other than TRPA1.
One criteria of bona fide mechanically-activated currents is that the current magnitude should be graded according to the stimulus magnitude 
. Indeed, we show here that increasing the stimulus magnitude increases the peak current amplitude for both SA and Transient currents. In order to estimate the maximum current amplitude, we applied graded mechanical stimuli of increasing intensity until the patch clamp seal became unstable. The average amplitude of the largest mechanically-evoked current was approximately 300 pA in both IB4 positive and IB4 negative small neurons from wild type mice of the TRPA1 strain. However in neurons from TRPA1−/− mice, the amplitude of the mechanical currents decreased in IB4 positive neurons and this decrease was due to a more than 60% reduction in the amplitude of the Transient currents.
Whereas Transient currents in IB4 positive neurons were reduced in TRPA1−/− neurons, acute inhibition of TRPA1 with HC-030031 in wild type neurons did not alter the amplitude of the mechanical-activated Transient currents in IB4 positive neurons. Thus, embryonic genetic ablation of the entire TRPA1 protein and acute pharmacological inhibition of TRPA1 channel function have different effects on the Transient mechanical current. One explanation may be that the TRPA1 protein is essential to the structure-function of a mechanically-sensitive complex that mediates the Transient current phenotype/profile, and without TRPA1 protein, neurons express an attenuated Transient current. Second, the complete absence of TRPA1 may result in downregulation of the expression of other mechanically-sensitive channels essential for the Transient current. This possibility is consistent with evidence that Transient mechanically-activated currents are mediated by Na+
ions, whereas TRPA1 is a non-selective cation channel 
. Third, the HC-030031 compound may fail to block mechanical activation of the TRPA1 channel mediating the Transient current in IB4 positive neurons. The site of action of HC-030031 on TRPA1 is not yet known (Magdalene Moran, personal communication), and the site(s) relevant for generation of mechanical currents may either be inaccessible to the compound or may themselves not be involved in the contribution of TRPA1 to Transient currents in IB4 positive neurons.
A definitive role of TRPA1 as a direct mechanically-gated ion channel could potentially be established through mechanical stimulation of heterologous cells expressing TRPA1. To this end, we expressed rat TRPA1 in HEK293 cells and found that although there was a trend for more mechanically-sensitive cells, the difference was not statistically significant and >50% of the TRPA1-transfected cells remained insensitive to mechanical stimuli. Furthermore, the resting membrane potential was significantly more depolarized in TRPA1-transfected cells, suggesting that the exogenous TRPA1 may have been constitutively active, resulting in a depolarized resting membrane potential. The finding that over half of the TRPA1-transfected cells were not mechanically sensitive suggests that TRPA1 alone is not sufficient to confer novel mechanical sensitivity to a cell and that associated proteins present in native neurons may be required to recapitulate mechanically-gated currents. Similarly, a recent study showed that overexpression of TRPA1 in neuroblastoma cell lines also does not confer novel mechanical sensitivity or induce any changes in endogenous mechanically-evoked currents in these heterologous cells 
An important consideration is that the function of an ion channel may be dependent on or heavily influenced by its native environment. The plasma membrane composition of HEK293 cells may be very different than that of DRG neurons. In native sensory neurons, TRPA1 is likely part of a macromolecular complex where it may interact intimately with other transmembrane proteins. For example, in some C fiber sensory neurons, TRPA1 may form heterotetramers with TRPV1, and the heterotetramers may have different functions than TRPA1 or TRPV1 homomers 
. Therefore, co-expression of other proteins, and possibly native sensory membrane phospholipids, may be essential to reconstitute “native” TRPA1 mechanical function in heterologous systems 
. Furthermore, TRPA1 may be downstream to the actual mechanotransducer in the sensory membrane. For example, a different ion channel may open during mechanical stimulation, resulting in an influx of Ca2+
that then activates TRPA1 
In summary, our parallel genetic and pharmacological data indicate that TRPA1 is one of the ion channels that underlie mechanically-activated currents at the plasma membrane of sensory neurons. Specifically, TRPA1 mediates the Slowly Adapting mechanical current in IB4 negative small-diameter sensory neurons, and many of these are peptide-containing, NGF-dependent C fiber nociceptors.