Humans rely on our sense of touch for a broad range of essential behaviors, such as
feeding, successful child rearing, and avoiding bodily harm. Although widely regarded
as one of the five basic senses, touch is a complex sense that encompasses numerous
modalities, including stretch, pressure, and vibration. Touch-sensitive neurons display
a corresponding diversity of force sensitivities, physiological outputs, and cellular
morphologies. Although forward genetic screens have identified several essential molecules
in invertebrate mechanosensory neurons, we are only now beginning to uncover molecular
players that govern the unique functions of discrete populations of touch receptors
in mammals. Recent progress has resulted from the convergence of mouse genetics, genomics,
developmental neurobiology, in vitro approaches, and neurophysiological techniques.
With this tool kit, we are now poised to answer long-standing questions: Do distinct
molecules transduce force in light-touch and pain receptors? What cell types and circuits
subserve different perceptual qualities in tactile discrimination? This Perspective
describes the most recent advances in our knowledge of molecules, cells, and circuits
that encode tactile stimuli, which will help uncover the mechanisms governing touch
transduction in mammals.
Our somatic senses of touch and pain enable numerous behaviors fundamental to human
existence, allowing us to eat, communicate, and survive. Acute pain is a warning signal
that alerts us to noxious mechanical, chemical, and thermal stimuli, which are potentially
tissue damaging. During inflammation or injury, we experience a heightened sensitivity
to touch that encourages us to protect the injured site. Despite this essential protective
function, pain can outlast its usefulness and become chronic. Numerous pathophysiological
conditions result in the chronic dysregulation of mechanosensory signaling, leading
to pain triggered by light touch (allodynia), as well as enhanced sensitivity to noxious
mechanical stimuli (hyperalgesia) (Gilron, 2006).
Light-touch receptors, which mediate discriminative touch, enable fine tactile acuity
that allows us to manipulate objects with high precision. As humans, we depend on
this skill for everyday tasks that range from eating with utensils to texting. Discriminative
touch is also central to social interactions, such as mating, maternal bonding, and
successful child rearing (Tessier et al., 1998; Feldman et al., 2010). Indeed, proper
brain development requires input from peripheral touch receptors (Fox, 2002). Depriving
infants of mechanosensory stimulation leads to striking developmental and cognitive
deficits (Kaffman and Meaney, 2007). For example, premature human infants housed in
incubators display delayed neurological development and growth, which can be improved
by only 45 min of touch a day (Ardiel and Rankin, 2010). In touch-deprived rodents,
attentional and behavioral deficits persist through adulthood, underscoring the importance
of mechanosensory inputs during development (Ardiel and Rankin, 2010).
To understand the senses of touch and pain, we must unravel peripheral mechanisms
that encode tactile stimuli and discover how the brain interprets these signals to
dictate behavior. The transduction of a physical force on the skin into an electrical
signal is the first step in the encoding of tactile stimuli. In this Perspective,
we focus on the most current developments in our understanding of the cells and molecules
that mediate touch transduction in the periphery. We refer the reader to recent reviews
that more comprehensively cover principles of mechanotransduction and somatosensory
signaling (Kung, 2005; Basbaum et al., 2009; Chalfie, 2009; Lumpkin et al., 2010).
Mammalian touch receptors are diverse
The skin is innervated by a variety of somatosensory neurons with distinct morphological
end-organs and physiological properties (Fig. 1). This array of cutaneous neuronal
subtypes is thought to represent different tactile qualities, such as shape, texture,
and vibration (Johnson, 2001), as well as a wide range of noxious stimuli (Basbaum
et al., 2009). With few exceptions, the correspondence between an end-organ and its
physiological response is only correlative, and class-specific molecular markers are
just now beginning to emerge (Loewenstein and Rathkamp, 1958; Woodbury and Koerber,
2007; Bourane et al., 2009; Luo et al., 2009).
Figure 1.
Cutaneous touch receptors. Mechanosensory afferents innervating mammalian skin display
morphological and functional diversity. Cartoons depict end-organs in hairy skin (left)
and glabrous skin (right), although innervation density is not meant to be representative.
For physiologically defined afferent classes, typical action potential trains evoked
by touch stimuli are schematized (center). Thickly myelinated Aβ afferents (blue shades)
are touch receptors that display RA or SA responses to mechanical stimuli. RA afferents
innervate hair follicles, Pacinian corpuscles, and Meissner’s corpuscles. SAI afferents
innervate epidermal Merkel cells (yellow), and SAII afferents are thought to innervate
Ruffini endings. Thinly myelinated Aδ afferents (green shades) include down-hair afferents
and A-mechanonociceptors. C-afferents (red and magenta), which surround hair follicles
(Park et al., 2003) and abundantly innervate the epidermis, include peptidergic nociceptors,
nonpeptidergic nociceptors, and C low-threshold mechanoreceptors.
Each somatosensory neuron has a soma located in the trigeminal ganglia or dorsal root
ganglia (DRG) and a branching sensory afferent that sends signals from the periphery
to the spinal cord and/or hindbrain. The peripheral branches of touch-receptive afferents
innervate the skin, where they transduce mechanical stimuli into action potentials.
These cutaneous sensory neurons can be physiologically classified based on conduction
velocity (set by degree of myelination), mechanical threshold, adaptation properties,
and modality, defined as the type of stimulus to which they best respond.
In general, light-touch receptors are thickly myelinated Aβ or thinly myelinated Aδ
afferents. These somatosensory neurons tend to have large somatal diameters and express
neurofilament 200, an intermediate filament protein. Within this broad category, rapidly
adapting (RA) and slowly adapting (SA) receptors can be distinguished.
RA afferents, which fire action potentials selectively at the onset and offset of
a touch stimulus, innervate several different cutaneous structures (Fig. 1). In the
hairy skin covering most of our body, lanceolate endings and circumferential afferents
surround hair follicles, where they are thought to signal hair movements. Notably,
down-hair afferents are Aδ fibers that are among the most sensitive light-touch receptors
in mammalian skin. In the glabrous skin of our palms, RA afferents innervate Pacinian
corpuscles and Meissner’s corpuscles, which are vibration receptors that encode texture.
The lamellae of these corpuscles serve as mechanical filters to set the adaptation
profiles of the Aβ afferents they envelope (Loewenstein and Mendelson, 1965).
SA afferents fire action potentials throughout a sustained touch stimulus (Fig. 1).
SAI afferents, which have the highest spatial acuity of mammalian touch receptors,
are proposed to represent object features such as edges and curvature (Johnson, 2001).
These Aβ afferents innervate Merkel cells (Woodbury and Koerber, 2007), which are
keratinocyte-derived epidermal cells that are required for SAI responses (Maricich
et al., 2009; Morrison et al., 2009; Van Keymeulen et al., 2009). SAII afferents,
which are sensitive to directional skin stretch, are thought to contribute to hand
grip and awareness of finger position (Johnson, 2001; Zimmermann et al., 2009). These
Aβ afferents are proposed to terminate in Ruffini endings, although the presence of
this end-organ in different species and skin areas is debated. Along with Aβ afferents,
the hairy skin is innervated by a rare subset of unmyelinated C-afferents that are
activated by innocuous touch stimuli and are marked by selective expression of vesicular
glutamate transporter 3 (Seal et al., 2009).
Nociceptors, which initiate pain perception, are thought to be free nerve endings
that fall into C-afferent or Aδ-afferent categories. A large variety of biochemically
and physiologically distinct C-afferent subtypes respond to an array of mechanical
and thermal stimuli, as well as endogenous and exogenous chemicals (Basbaum et al.,
2009). In many cases, nociceptors are polymodal, responding robustly to multiple sensory
stimuli. Although most C-afferents have traditionally been classified as nociceptors,
based on their high mechanical thresholds and projection patterns to the spinal cord
(Smith and Lewin, 2009), recent studies have implicated C-afferents in other cutaneous
senses, such as warm and cool (Peier et al., 2002; Dhaka et al., 2008). High-threshold
A-afferent mechanonociceptors are also observed electrophysiologically, although the
cutaneous end-organs of these afferents are not known (Zimmermann et al., 2009).
C-afferents richly innervate the epidermis of hairy and glabrous skin (Fig. 1). Peptidergic
afferents, which express neuropeptides such as Substance P or calcitonin gene-related
peptide, innervate mid-layers of the epidermis. In contrast, nonpeptidergic afferents,
most of which express the Mas-related G protein–coupled receptor MrgD, selectively
innervate the outermost living skin layer (stratum granulosum) (Zylka et al., 2005).
Interestingly, under normal and inflammatory conditions, mice lacking MrgD-positive
afferents display decreased responsiveness to noxious mechanical stimuli but normal
sensitivity to heat and cold (Cavanaugh et al., 2009). Thus, these afferents may play
a selective role in acute mechanical pain and tactile hypersensitivity.
An intriguing open question is whether cutaneous afferents themselves mediate transduction
in all mechanosensory modalities or whether epidermal cells also play a role in sensory
signaling (Lumpkin and Caterina, 2007). It is clear that nociceptors express some
sensory transduction channels, such as the capsaicin receptor transient receptor potential
vanilloid (TRPV)1 (Caterina et al., 1997); however, keratinocytes also express putative
sensory transduction channels, including TRPV3 and TRPV4 (Lumpkin and Caterina, 2007).
Moreover, keratinocytes, Merkel cells, and Pacinian corpuscles express neurotransmitters
(Lumpkin et al., 2010), receptors for which are expressed in somatosensory afferents.
For example, keratinocytes release ATP in response to sensory stimuli in vitro, and
MrgD-positive epidermal sensory neurons express the ATP-gated ion channel P2X3 (Dussor
et al., 2008). Although these findings are suggestive, the roles of epidermal cells
in touch transduction have not been defined.
Molecular specification of somatosensory cell types
Developmental studies, particularly in genetically modified mouse models, have begun
to illuminate mechanisms underpinning the variety of mammalian touch receptors (Luo
et al., 2007). Almost all nociceptors require nerve growth factor and its receptor
TrkA for specification. At late embryonic stages, nonpeptidergic C-afferents begin
to express the transcription factor Runx1 and Ret, a receptor for glial-derived neurotrophic
factor ligands (Kramer et al., 2006; Luo et al., 2007). Postnatally, these nonpeptidergic
nociceptors turn off TrkA expression, whereas peptidergic C-afferents maintain TrkA
expression and require nerve growth factor for survival.
Touch receptors are also specified by neurotrophic factors and developmental transcription
factors. RA afferents depend on early embryonic Ret expression and the transcription
factor MafA for proper development (Bourane et al., 2009; Luo et al., 2009). Down-hair
lanceolate endings are distinguished by their developmental dependence on neurotrophin
(NT)-4 (Stucky et al., 1998); Merkel cell–neurite complexes generally require NT-3
and its receptor TrkC for postnatal survival (Airaksinen et al., 1996). In whisker
follicles, Merkel cell innervation depends on the transcription factor Runx3 (Senzaki
et al., 2010). Proprioceptive neurons, which represent another NT-3–dependent mechanosensory
population, are also lost in Runx3 mutants (Levanon et al., 2002; Kramer et al., 2006).
Like mechanosensory hair cells of the inner ear, epidermal Merkel cells are vertebrate-specific
cells whose development depends on the transcription factor Atonal 1 (Maricich et
al., 2009; Morrison et al., 2009; Van Keymeulen et al., 2009).
Based largely on their distinct developmental pathways, some types of touch receptors
can now be identified with genetically encoded markers (Lumpkin et al., 2010). These
markers are essential tools for identifying molecules that govern the distinct responses
of touch-receptor subtypes.
Molecular mechanisms of mammalian touch transduction
In mechanosensory cells, ion channels underlie the transduction of mechanical stimuli
into electrical signals. There are two models of how such ion channels are activated.
The first model postulates that force-sensitive ion channels are directly activated
by changes in membrane tension or distortion. This is the case for the osmosensitive
bacterial channels MscS and MscL (Kung, 2005) and members of the two-pore potassium
channel family, KCNK (Kung et al., 2010). The second model posits that gating requires
tethering molecules that link the transduction channel to the cytoskeleton or extracellular
matrix. This model stems from studies in mechanosensory hair cells, where cadherin
family proteins and myosins are required for mechanotransduction (Schwander et al.,
2010); however, the molecular basis of mechanotransduction in mammalian somatosensory
neurons remains enigmatic.
Members of the TRP channel, acid-sensing ion channel, and KCNK channel families have
been proposed to function as transduction channels in somatosensory neurons. Because
genetic deletion of candidates only subtly alters cellular and/or behavioral mechanosensitivity,
the importance of these channels in mammalian mechanotransduction remains controversial.
These issues have been extensively discussed in several reviews and will not be covered
in detail here (Lewin and Moshourab, 2004; Christensen and Corey, 2007; Lumpkin and
Caterina, 2007; Basbaum et al., 2009). More recently, two members of the FAM38 gene
family, FAM38A and FAM38B, have been implicated in somatosensory mechanotransduction.
A role for FAM38A and FAM38B in mechanotransduction stems from an unbiased screen
to identify genes required for mechanosensitivity in the Neuro2A mouse neuroblastoma
cell line (Coste et al., 2010). Each gene is a complex locus predicted to produce
more than a dozen isoforms through alternative promoters and splicing (Thierry-Mieg
and Thierry-Mieg, 2006). The proteins encoded by these genes, Piezo1 and Piezo2, are
large membrane proteins with up to 30 and 34 predicted transmembrane domains, respectively
(Fig. 2 A); however, no putative pore domains or channel-like repetitive domains have
been identified. Piezo1 is broadly expressed, including in mechanosensitive tissues
such as bladder, lung, and skin (Thierry-Mieg and Thierry-Mieg, 2006; Coste et al.,
2010). Piezo1 is also expressed in senile plaque–associated astrocytes (Satoh et al.,
2006). Piezo2 transcripts are also detected in several tissues but appear to be most
abundant in DRG, bladder, and lung (Thierry-Mieg and Thierry-Mieg, 2006; Coste et
al., 2010).
Figure 2.
Piezo1 and Piezo2 are candidate mechanotransduction molecules. (A) Predicted hydropathy
plots for Piezo1 and Piezo2 proteins. The plot displays putative transmembrane (red),
intracellular (black), and extracellular (gray) domains, as predicted by the TMHMM
2.0 server. (B) Mechanically activated currents in HEK293T cells expressing Piezo1
(FAM38A; left) or Piezo2 (FAM38B; right). Representative inward currents in response
to a series of 1-µm mechanical steps applied via a glass probe. Whole cell recordings
performed at −80 mV. B is modified with permission from Coste et al. (2010).
Notably, Piezo1 was also identified in a functional screen for transcripts that regulate
integrins, which are mechanosensitive cell adhesion molecules (McHugh et al., 2010).
Integrins are transmembrane receptors that serve as a mechanical link between the
extracellular matrix and the cytoskeleton. They serve as signaling hubs that, in response
to mechanical load, initiate numerous intracellular signaling cascades that govern
gene transcription, cell motility, and differentiation (Legate et al., 2009). Integrins
mediate mechanotransduction in a variety of physiological contexts, including cell
rigidity, migration, organogenesis, and development. FAM38A was shown to activate
integrin signaling by recruiting the R-Ras GTPase to the ER. Whether Piezo1 and Piezo2
are functional ion channels, accessory subunits of mechanosensitive channels, or signaling
molecules within a mechanosensitive pathway (e.g., integrin signaling) remains unanswered.
In favor of a channel hypothesis, however, expression of Piezo1 or Piezo2 confers
displacement- and suction-evoked currents in heterologous cells, such as HEK293 cells
(Fig. 2 B).
The diversity of candidate transduction channels raises an important question: what
criteria must be satisfied by a bona fide mechanotransduction channel in mammalian
somatosensory neurons? Christensen and Corey (2007) previously outlined a set of functional
criteria for assessing whether a candidate ion channel is directly activated by mechanical
stimuli. Here, we extend this set of criteria to assess whether a candidate mediates
mammalian somatosensory mechanotransduction, using Piezo1 and Piezo2 as examples.
Most studies of previous transduction candidates used different stimuli and criteria
to assess mechanosensitivity, thus making it difficult to compare between studies.
Is the candidate in the right place?
It is possible that distinct molecules transduce mechanical stimuli in the different
classes of touch receptors schematized in Fig. 1. Thus, at a minimum, a candidate
transduction molecule must be expressed in the skin or sensory ganglia and localize
to at least one sensory cell type. Because transduction occurs in cutaneous end-organs,
bone fide transduction channels should also localize to the plasma membranes of peripheral
endings. It is worth noting that a candidate need not be highly expressed to function
as a transduction channel, especially if it mediates transduction in only a small
population of touch-sensitive neurons.
How well do the Piezos meet these expression criteria? Quantitative PCR analysis shows
preferential expression of Piezo2 in somatosensory ganglia and Piezo1 enrichment in
the skin. In situ hybridization shows Piezo2 localization in ∼20% of DRG neurons.
Most of these are likely to represent nociceptors, as they coexpress nociceptive markers
such as peripherin or TRPV1. Other Piezo2-positive DRG neurons express the myelination
marker NF200; these Aβ or Aδ neurons might include light-touch receptors (Coste et
al., 2010). Antibody staining of heterologously expressed Piezo2 shows high intracellular
levels and, to a lesser extent, plasma membrane expression (Coste et al., 2010; McHugh
et al., 2010). Similarly, a GFP-tagged Piezo1 localizes to the ER in HeLa cells (McHugh
et al., 2010). The subcellular distribution of endogenously expressed Piezo1 or Piezo2
in the skin or DRG neurons has not yet been reported. Thus, the tissue distribution
is consistent with a role for Piezos in mechanotransduction, but key information about
subcellular localization is still lacking. Moreover, because Piezo2 is expressed in
only a subset of DRG neurons, additional candidates must be identified in other somatosensory
cell types.
Is the candidate intrinsically mechanosensitive?
If an ion channel is directly gated by force, a candidate’s mechanical properties
can be directly compared with endogenous transduction mechanisms. One caveat is that
heterologous expression will not produce mechanosensitive currents if accessory proteins
or specific cellular contexts are required for force gating. Indeed, the Deg/ENaC
isoforms that transduce gentle touch in Caenorhabditis elegans do not appear to be
mechanically gated when heterologously expressed (Lumpkin et al., 2010). This stumbling
block has made it difficult to assess mammalian Deg/ENaC mechanotransduction candidates,
such as the acid-sensing ion channels, that do not confer mechanosensitivity in heterologous
cells. For such ion channels, we must rely on other physiological properties, such
as selectivity or pharmacological profiles, for comparison with endogenous currents.
Like the mechanosensitive KCNK channels (Kung et al., 2010), either Piezo1 or Piezo2
expression alone is sufficient to confer mechanically evoked currents in heterologous
cell types (Coste et al., 2010). This finding is promising because the mechanosensitivity,
pharmacology, and biophysical characteristics of Piezo-dependent currents can now
be directly compared with those of endogenous mechanically activated currents in sensory
neurons.
Does the candidate display characteristics of endogenous transduction channels in
sensory neurons?
Somatosensory neurons retain mechanosensitivity when dissociated and placed in culture.
Because it is not clear which in vitro mechanical stimuli best represents tactile
stimulation in vivo, a variety of mechanical stimulus paradigms have been tested on
dissociated sensory neurons. Several of these paradigms reliably produce mechanosensitive
responses in sensory neurons; however, their relation to physiological forces in tissues
remains unclear. Nonetheless, in vitro recordings are, at present, the most direct
way to assess mechanically evoked responses at the cellular level.
Hypo-osmotic solutions induce cell swelling that leads to calcium influx and neuronal
excitation in a subset of sensory neurons (Fig. 3 A; Viana et al., 2001). Osmotic
responses require extracellular calcium but are not significantly blocked by voltage-activated
calcium channel antagonists, suggesting that swelling triggers calcium influx through
an unknown conductance. A second stimulus paradigm is radial stretch of neurons cultured
on elastic membranes. Like osmotic stimuli, radial stretch triggers calcium increases
in a subset of sensory neurons that require extracellular calcium and are not inhibited
by voltage-activated calcium channel blockers (Fig. 3 B; Bhattacharya et al., 2008).
Third, like many mammalian cell types, cultured sensory neurons have stretch-activated
channels that are gated by suction or pressure applied through a recording pipette
(see, for example, Cho et al., 2006). The fourth and most commonly used technique
for probing cellular mechanosensitivity is focal displacement applied to the soma
or neurite (Fig. 3 C). Such stimulation triggers calcium influx and several currents
with distinct properties.
Figure 3.
Cell-based assays to probe mechanotransduction. (A) Application of hypo-osmotic solutions
causes stretch-evoked calcium signals in DRG neurons. (B) Radial stretch of DRG neurons
grown on silastic membranes elicits dose-dependent calcium influx. (C) Membrane suction
activates stretch-activated channels while focal pressure applied to the DRG soma
triggers calcium influx in cultured DRG neurons. (D) Focal pressure applied to the
neurites of sensory neurons elicits RA, IA, and SA currents. D is modified with permission
from Lechner et al. (2009. EMBO J. 28:1479–1491).
Several groups have reported displacement-evoked mechanosensitive currents; however,
stimulation protocols, recording conditions, parameters measured, and model organism
vary between these reports. As such, basic properties of mechanically evoked currents
differ somewhat between studies, making it difficult to define benchmarks for comparison
with candidate mechanotransduction channels. Touch-evoked currents can be elicited
by 2–16-µm displacements of cell somata (McCarter et al., 1999; Drew et al., 2002,
2007; Drew and Wood, 2007; Hao and Delmas, 2010; Rugiero et al., 2010) or <1-µm displacement
of neurites (Hu and Lewin, 2006). One commonality is the existence of three types
of displacement-evoked currents: RA, intermediate adapting (IA), and SA (Table I and
Fig. 3 C, bottom).
Table I.
Properties of mechanically activated currents in cultured sensory neurons and HEK293
cells expressing Piezo1 or Piezo2
Cell type
Ionic selectivity
Stimuli
Activation tau
Inactivation tau
Adaptation mechanism
Block
µm
ms
ms
Rat DRG RAa
b
c
d
e
f
Nonselective cation
2–12a
ND
3
Voltage-dep., Ca2+-indep.
Ca2+, Gd3+, NMB1, ruthenium red, FM1-43, cytochalasin B
Rat DRG IAd
e
Nonselective cation
2–12
ND
21
ND
NMB1
Rat DRG SAa
b
c
d
e
f
Nonselective cation
2–12
ND
296 (τ1)1,140 (τ2)
Voltage-dep., Ca2+-indep.
Gd3+, ruthenium red, cytochalasin B, Ca2+, FM1-43
Mouse DRG RAg
h
Sodiumg
0.75–1 (neurite) 4–6 (soma)
0.8–1.0
1.05–1.92
ND
Gd3+, NMDG
Nonselective cationh
16
3
47–57
Ruthenium red
Mouse DRG IAg
ND
0.75–1 (neurite) 4–6 (soma)
0.5–0.7
17–26
ND
Gd3+
Mouse DRG SAg
i
Nonselective cation
0.75–1 (neurite) 4–6 (soma)
0.4–1.3
>230
ND
Gd3+, ruthenium red, HC030031i
HEK293 Piezo1j
Nonselective cationj
3
ND
17
ND
Ruthenium red Gd3+, Ca2+
HEK293 Piezo2j
Nonselective cationj
ND
ND
7
ND
Ruthenium red Gd3+, NMDG
a
McCarter et al. (1999).
b
Drew et al. (2002).
c
Drew and Wood (2007).
d
Drew et al. (2007).
e
Hao and Delmas (2010).
f
Rugiero et al. (2010).
g
Hu and Lewin (2006).
h
Drew et al. (2004).
i
Vilceanu and Stucky (2010).
j
Coste et al. (2010).
RA currents display fast kinetics of activation and desensitization (0.5 and 1 ms,
respectively) and block by several pharmacological agents. In rat neurons, nonselective
RA cation currents are blocked by: Ca2+; Gd3+, which inhibits many types of stretch-activated
ion channels; the conotoxin NMB1; the styryl dye FM1-43; the TRP channel inhibitor
ruthenium red; and cytochalasin B, which inhibits actin polymerization (McCarter et
al., 1999; Drew et al., 2002, 2007; Drew and Wood, 2007; Hao and Delmas, 2010; Rugiero
et al., 2010). Mouse neurons display different characteristics in different studies.
In one study, a 16-µm displacement of cell somata elicited ruthenium red–sensitive
nonselective cation currents (Drew et al., 2004). In other studies, ≤1-µm displacements
elicited sodium-selective currents blocked by Gd3+ and NMDG (Hu and Lewin, 2006; Lechner
et al., 2009).
At least two populations of RA neurons can be distinguished by their action potential
shapes and the developmental stage at which they appear (Lechner et al., 2009). At
mouse E13.5, a subset of large DRG neurons, which express TrkB or TrkC, display mechanosensitive
RA currents that are Na+ selective. These neurons are likely to correspond to low-threshold
Aβ afferents, such as light-touch receptors and proprioceptors. At E15.5, a second
population of RA neurons appears that have small somatal diameters and broad action
potentials characteristic of nociceptors.
Other classes of putative nociceptors display IA and SA mechanically evoked currents
in culture (Hu and Lewin, 2006). IA currents are nonselective, inactivate in tens
of milliseconds, are blocked by NMB1 (rat) and Gd3+ (mouse), and are relatively rare
in embryonic neurons (Lechner et al., 2009). SA currents, which emerge postnatally
in dissociated sensory neurons (Lechner et al., 2009), are nonselective, inactivate
over hundreds of milliseconds, and are blocked by Gd3+, ruthenium red in mouse and
rat neurons, cytochalasin B, Ca2+, FM1-43 (rat neurons) (McCarter et al., 1999; Drew
et al., 2002, 2007; Drew and Wood, 2007; Hao and Delmas, 2010; Rugiero et al., 2010),
and the TRPA1 antagonist HC030031 in mouse neurons (Vilceanu and Stucky, 2010).
In cultured rat sensory neurons, mechanically evoked RA and SA currents display calcium-independent
and voltage-dependent desensitization during sustained mechanical stimuli (Hao and
Delmas, 2010; Rugiero et al., 2010). This is markedly different from the adaptation
properties of mechanosensitive currents measured in inner-ear hair cells, indicating
that distinct mechanisms regulate transduction in these mechanosensory cell types.
Mechanically evoked currents in Piezo1- or Piezo2-expressing HEK293T cells share several
features with displacement-evoked currents in sensory neurons. For example, similar
to currents in DRG neurons, Piezo-dependent currents are activated by micrometer focal
displacement of somata. Activation kinetics appear to be in the low millisecond range.
Piezo1 inactivation occurs with a time constant of 15 ms, most similar to IA currents
in sensory neurons. Piezo1 is blocked by Ca2+, Gd3+, and ruthenium red, whereas Piezo2
is blocked by NMDG, Gd3+, and ruthenium red. The spider toxin GsMTx-4, which inhibits
stretch-activated channels in a variety of cell types (Bowman et al., 2007), also
blocks Piezo1-depedent currents (Bae et al., 2011).
Piezo-dependent currents are also strikingly similar to suction-activated currents
observed in a subset of small-diameter cultured sensory neurons (Cho et al., 2006).
For example, the single-channel conductance of Piezo1-dependent channels in N2A cells
matches that of one class of endogenous stretch-activated channels (23 pS). Like Piezo-dependent
currents, these stretch-activated channels are Gd3+-sensitive nonselective cation
channels.
Collectively, these physiological features support a parsimonious model in which Piezo
proteins form mechanosensitive channels; however, many questions remain unanswered.
At a biophysical level, stimulus–response relations, adaptation properties, and activation/inactivation
kinetics need to be more thoroughly defined.
Is the candidate required for mechanotransduction in cultured sensory neurons?
A bone fide transduction channel must be required for native mechanotransduction currents.
Thus, gene disruption, RNA interference, or selective antagonists that target a candidate
should alter stimulus–response properties, adaptation, ionic selectivity, or conductance
of endogenous currents in sensory neurons. Functional disruption of a mechanotransduction
channel is also expected to be modality specific, altering only mechanical sensitivity
and not responsiveness to thermal or chemical stimuli. Promisingly, treatment of cultured
DRG neurons with Piezo2 short-interfering RNA decreased the proportion of sensory
neurons with RA currents and showed a trend toward increased incidence of mechanically
insensitive neurons (Coste et al., 2010). In contrast, the proportion of neurons exhibiting
IA or SA currents was comparable in control and Piezo2-targeted cultures. Collectively,
these findings suggest that Piezo2 is specifically required for mechanically evoked
RA currents in cultured sensory neurons.
Is the candidate required for touch-evoked responses in vivo?
An essential step in validating candidate transduction molecules is to use in vivo
approaches to determine the functional importance of the candidate in somatosensory
signaling. This is typically achieved by disrupting a candidate gene in mice and then
assaying somatosensory signaling with intact electrophysiological recordings and behavioral
assays.
Intact recordings provide key information by demonstrating whether a candidate is
required for touch sensitivity in specific subsets of touch receptors and whether
it functions in the periphery (Fig. 1). In rodents, mechanically evoked action potentials
can be recorded extracellularly from teased peripheral afferents in an ex vivo skin-saphenous
nerve preparation (for protocols see Zimmermann et al., 2009). This recording configuration
has been widely used to evaluate the importance of developmental pathways, transduction
candidates, and voltage-activated ion channels in peripheral sensory signaling. For
example, when compared with wild-type controls, mice lacking stomatin domain protein
SLP3 show a high proportion of mechanically insensitive cutaneous afferents in skin
nerve recordings (Wetzel et al., 2007). Responses can also be measured intracellularly
from DRG somata in vivo (Ma et al., 2010) or in an ex vivo skin–DRG preparation, which
allows a neuron’s peripheral end-organs and central projections to be visualized with
neuronal tracers (Woodbury and Koerber, 2007; Seal et al., 2009). This is an important
advantage because it can be used to identify the morphology of touch receptors that
are classified by their physiological properties (Woodbury and Koerber, 2007).
Two standard behavioral assays are used to measure touch sensitivity in rodents. First,
calibrated von Frey filaments are used to apply force to the plantar surface of a
mouse hind paw, and either the force required to elicit paw withdrawal or the number
of withdrawal responses to a given force is recorded (Chaplan et al., 1994). Second,
the Randall–Selitto test uses a clamping device that applies progressively higher
pressure on the tail (or hind paw) until the rodent withdraws, at which point force
magnitude is measured (Randall and Selitto, 1957). Although these tests are robust
for probing function of pain-sensing nociceptors, they are not designed to analyze
the wide array of sensory neurons that mediate discriminative touch.
New behavioral assays are needed to assess sensitivity to light touch, texture, and
vibration. One such assay is a two-choice preference-based tactile acuity test, whereby
mice prefer exploring textured floor gratings over smooth surfaces (Wetzel et al.,
2007). Mice lacking SLP3 display altered texture preferences, suggesting that SLP3
is required for normal responses to light-touch stimuli (Wetzel et al., 2007). The
use of mice that lack specific subsets of sensory afferent types will greatly facilitate
the design of new behavioral assays that are fine-tuned to a specific class of mechanoreceptor
(Bourane et al., 2009; Luo et al., 2009; Maricich et al., 2009).
Does the candidate gene encode a pore-forming transduction channel?
When a transduction channel candidate is shown to be necessary and/or sufficient for
mechanically evoked currents, a key remaining validation step is to determine whether
the gene encodes a pore-forming ion channel. This is critical for defining mechanosensory
mechanisms because, rather than functioning as a transduction channel, a candidate
might be required for proper expression, trafficking, or gating of transduction channels.
For example, Piezo1 might play a role in regulating the store-operated calcium channel
Orai (Wu et al., 2007). FAM38A knockdown in HeLa cells decreases calcium release from
intracellular stores and attenuates calcium influx through Orai channels (McHugh et
al., 2010). Although the authors speculate that Piezo1 might function as an ER calcium
release channel, it is equally plausible that it modulates Orai function, which is
responsible for calcium influx and the refilling of calcium stores; any decrease in
Orai activity would lead to smaller stores and diminished influx, as observed. Thus,
future experiments are needed to determine whether Piezos are pore-forming channels
or channel modulators, as the approaches outlined above cannot distinguish between
these possibilities.
Protein engineering offers ingenious methods for demonstrating that a gene encodes
a pore-forming transduction channel. Point mutations can be engineered to confer pharmacological
sensitivity or to cause signature alterations in specific biophysical properties,
such as ionic selectivity or conductance (O’Hagan et al., 2005). These mutant isoforms
can then be used to test for functional rescue of candidate gene disruption in mechanosensory
neurons. If the gene encodes a bona fide transduction channel, endogenous mechanically
evoked currents should display the pharmacological or biophysical signature of the
point mutant after rescue. This approach has been used to successfully validate Deg/ENaC
subunits and TRP-4 as mechanotransduction channels in C. elegans and myosins as adaptation
motors in mechanosensory hair cells (Holt et al., 2002; O’Hagan et al., 2005; Kang
et al., 2010).
Because Piezo proteins lack sequence similarity to all known ion channels, implementing
this strategy is likely to require extensive structure–function analysis to identify
putative pore regions, to define signature point mutations, and to confirm that these
mutations do not alter protein trafficking or subcellular localization. The discovery
that Piezo genes induce robust mechanosensitive currents in many cell types makes
this powerful approach possible.
Conclusions
Among sensory systems, the molecular mechanisms underlying touch remain most enigmatic.
Based on studies in cultured sensory neurons and heterologous systems, Piezos are
promising new candidates for mediating mechanotransduction; however, key studies are
needed to understand the nature of these molecules and the roles they play in somatosensation
and other mechanosensitive cell types. As described above, the critical experiments
needed to demonstrate a requirement for Piezo proteins in cutaneous somatosensory
transduction include showing an altered tactile phenotype in Piezo-deficient mice
and proving that Piezo isoforms contribute to a pore-forming channel in vivo. In addition,
as new tools for probing touch in vitro and in vivo become available, other candidate
molecules must also be revisited and new candidates remain to be discovered. Only
by defining the biophysical and pharmacological signatures for each subtype of sensory
neuron, and matching behavioral output to each subtype, can we understand the complex
mechanisms underlying our sense of touch.
This Perspectives series includes articles by Farley and Sampath, Schwartz and Rieke,
Reisert and Zhao, and Zhang et al.