Our findings elucidate the molecular mechanism through which pungent agents of Xanthoxylum
plants mediate their unique psychophysical effects, akin to the experience of touching one’s tongue to the terminals of a 9-V battery. This sensation is rather distinct from that elicited by other pungent natural products, such as those derived from chili peppers or wasabi, which produce a more acute and painful irritation that is also associated with local tissue inflammation and pain hypersensitivity. Moreover, our data suggest that sanshool is unique among pungent agents in that its excitatory actions are mediated by inhibition of pH-sensitive background potassiumchannels, rather than through the more familiar mechanism involving direct activation of an excitatory TRP channel2
. Indeed, such functional differences may contribute to perceived differences in the pungency (onset or intensity) of these natural-product irritants. More broadly, the mechanism of sanshool-evoked K+
channel inhibition differs from that of most chemosensory agents, which involve G protein–coupled receptor signaling pathways35,36
Differences in pungency perception and other neurally mediated effects may also reflect the activation of overlapping, but distinct, subpopulations of primary afferent sensory neurons. For example, capsaicin activates a subgroup of small-diameter sensory neurons, of which approximately 50% are also activated by mustard oil. This latter component of dually responsive neurons also express proinflammatory neuropeptides (CGRP and substance P), accounting for both the acute pain and the neurogenic inflammatory actions commonly associated with exposure to chili peppers, wasabi or garlic. In contrast, we show that sanshool excites only the subset of capsaicin-sensitive neurons that are mustard oil insensitive, thereby excluding most of the peptidergic nociceptors. This is consistent with the fact that exposure to sanshool is not associated with intense pain, neurogenic inflammation or hyperalgesia.
We also found that sanshool activates large-diameter, TrkC-positive, myelinated neurons, which are generally associated with proprioception and the detection of non-noxious mechanical stimuli, such as light touch or vibration37
. It is therefore notable that the majority of TrkC-positive, osmotically sensitive neurons are also sensitive to sanshool, consistent with the idea that sanshool elicits its tingling and buzzing sensation by activating a cohort of touch-sensitive fibers. Sanshool therefore serves as the first pungent natural product with which to identify this specific cohort of mechanosensitive primary afferent neurons.
At the molecular level, sanshool has been proposed to activate neurons by opening excitatory ion channels9,11,12
, and we were therefore surprised to find that it depolarizes neurons by inhibiting background potassium channels. Aside from our direct electrophysiological characterization of sanshool-sensitive membrane currents in sensory neurons, our examination of various cloned channels also rules out a significant involvement of members of the TRP channel family that have been suggested to play a role in this response, including receptors for capsaicin, mustard oil and menthol (TRPV1, TRPA1 and TRPM8, respectively). Rather, our data demonstrate that a subset of pH-sensitive two-pore K+
channels, namely KCNK3, KCNK9 and KCNK18, are the molecular targets of sanshool action. Indeed, sanshool now provides a new pharmacological tool for discriminating among two-pore K+
channel subtypes, one that is more selective in its action compared to rutheniumred, zinc, protons and anesthetics, all of which target multiple KCNK subtypes as well as other ion channels.
Our data clearly show that KCNK3 and KCNK18 are the principal sanshool-sensitive subtypes in sensory neurons, whereas in CGNs, KCNK3 and KCNK9 predominate, thereby accounting for the bulk of cellular sensitivity to sanshool. Our pharmacological results with hydroxy-β-sanshool indicate that KCNK3 homomeric channels are not primary contributors to the excitatory effects of sanshool, where KCNK18 or KCNK3/KCNK9 heteromeric complexes must account for these actions in sensory neurons or CGNs, respectively. Further analysis of the relative contributions of these channels to sanshool sensitivity must await the development of additional subtype-selective antagonists or the generation of triple KCNK3-, KCNK9- and KCNK18-deficient mice, because animals lacking any single KCNK subtype show compensatory upregulation of related channels31,38
. Similarly, the analysis of such animals will show whether additional ion channels or receptors contribute to sanshool sensitivity at the behavioral level.
plants have been exploited for centuries as natural analgesics to alleviate acute and chronic pain. Fruits of these plants have also been use extensively in the kitchen because of their unique pungent qualities. In contrast to the familiar burning or irritating pain elicited by chili peppers or mustard extracts, the sensorial experience produced by Szechuan peppercorns is more generally described as “tingling and numbing,” “mild electric shock” or a “pins and needles” effect9,11
. These psychophysical percepts are in many ways consistent with the cellular and molecular sites that we identify as sanshool targets. Thus, the numbing qualities of sanshoolmay resemble the effects of anesthetics on KCNK3, KCNK9 and KCNK18. The tactile component may result from the activation of large-diameter, touch-sensitive fibers, whereas the pungent or irritant qualities may involve excitation of nonpeptidergic, capsaicin-sensitive nociceptors. The identification of sanshool-sensitive channels and sensory neuron subtypes represents an essential first step in understanding mechanisms underlying this unique pungency and its relationship to tactile sensitivity.