Rui B. Chang

The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions.

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca2+ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca2+ on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca2+ permeability was greatly reduced, extracellular Ca2+ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca2+ buffering, which allowed intracellular Ca2+ levels to become elevated upon entry through D918A channels. Application of Ca2+ to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca2+ by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg2+, Ba2+, and Ca2+ but potentiated only by Ba2+ and Ca2+. Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca2+-binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca2+ influx through TRPA1 to nociceptive signaling.

TRPA1 Is a Component of the Nociceptive Response to CO2

In humans, high concentrations of CO2, as found in carbonated beverages, evoke a mixture of sensations that include a stinging or pungent quality. The stinging sensation is thought to originate with the activation of nociceptors, which innervate the respiratory, nasal, and oral epithelia. The molecular basis for this sensation is unknown. Here we show that CO2 specifically activates a subpopulation of trigeminal neurons that express TRPA1, a mustard oil- and cinnamaldehyde-sensitive channel, and that these responses are dependent on a functional TRPA1 gene. TRPA1 is sufficient to mediate responses to CO2 as TRPA1 channels expressed in HEK-293 cells, but not TRPV1 channels, were activated by bath-applied CO2. CO2 can diffuse into cells and produce intracellular acidification, which could gate TRPA1 channels. Consistent with this mechanism, TRPA1 channels in excised patches were activated in a dose-dependent manner by intracellular protons. We conclude that TRPA1, by sensing intracellular acidification, constitutes an important component of the nociceptive response to CO2.

Synchronous evolution of an odor biosynthesis pathway and behavioral response.

BACKGROUND Rodents use olfactory cues for species-specific behaviors. For example, mice emit odors to attract mates of the same species, but not competitors of closely related species. This implies rapid evolution of olfactory signaling, although odors and chemosensory receptors involved are unknown. RESULTS Here, we identify a mouse chemosignal, trimethylamine, and its olfactory receptor, trace amine-associated receptor 5 (TAAR5), to be involved in species-specific social communication. Abundant (>1,000-fold increased) and sex-dependent trimethylamine production arose de novo along the Mus lineage after divergence from Mus caroli. The two-step trimethylamine biosynthesis pathway involves synergy between commensal microflora and a sex-dependent liver enzyme, flavin-containing monooxygenase 3 (FMO3), which oxidizes trimethylamine. One key evolutionary alteration in this pathway is the recent acquisition in Mus of male-specific Fmo3 gene repression. Coincident with its evolving biosynthesis, trimethylamine evokes species-specific behaviors, attracting mice, but repelling rats. Attraction to trimethylamine is abolished in TAAR5 knockout mice, and furthermore, attraction to mouse scent is impaired by enzymatic depletion of trimethylamine or TAAR5 knockout. CONCLUSIONS TAAR5 is an evolutionarily conserved olfactory receptor required for a species-specific behavior. Synchronized changes in odor biosynthesis pathways and odor-evoked behaviors could ensure species-appropriate social interactions.

A proton current drives action potentials in genetically identified sour taste cells

Five tastes have been identified, each of which is transduced by a separate set of taste cells. Of these sour, which is associated with acid stimuli, is the least understood. Genetic ablation experiments have established that sour is detected by a subset of taste cells that express the TRP channel PKD2L1 and its partner PKD1L3, however the mechanisms by which this subset of cells detects acids remain unclear. Previous efforts to understand sour taste transduction have been hindered because sour responsive cells represent only a small fraction of cells in a taste bud, and numerous ion channels with no role in sour sensing are sensitive to acidic pH. To identify acid-sensitive conductances unique to sour cells, we created genetically modified mice in which sour cells were marked by expression of YFP under the control of the PKD2L1 promoter. To measure responses to sour stimuli we developed a method in which suction electrode recording is combined with UV photolysis of NPE-caged proton. Using these methods, we report that responses to sour stimuli are not mediated by Na+ permeable channels as previously thought, but instead are mediated by a proton conductance specific to PKD2L1-expressing taste cells. This conductance is sufficient to drive action potential firing in response to acid stimuli, is enriched in the apical membrane of PKD2L1-expressing taste cells and is not affected by targeted deletion of the PKD1L3 gene. We conclude that, during sour transduction, protons enter through an apical proton conductance to directly depolarize the taste cell membrane.

A TRPA1-dependent mechanism for the pungent sensation of weak acids

Acetic acid produces an irritating sensation that can be attributed to activation of nociceptors within the trigeminal ganglion that innervate the nasal or oral cavities. These sensory neurons sense a diverse array of noxious agents in the environment, allowing animals to actively avoid tissue damage. Although receptor mechanisms have been identified for many noxious chemicals, the mechanisms by which animals detect weak acids, such as acetic acid, are less well understood. Weak acids are only partially dissociated at neutral pH and, as such, some can cross the cell membrane, acidifying the cell cytosol. The nociceptor ion channel TRPA1 is activated by CO2, through gating of the channel by intracellular protons, making it a candidate to more generally mediate sensory responses to weak acids. To test this possibility, we measured responses to weak acids from heterologously expressed TRPA1 channels and trigeminal neurons with patch clamp recording and Ca2+ microfluorometry. Our results show that heterologously expressed TRPA1 currents can be induced by a series of weak organic acids, including acetic, propionic, formic, and lactic acid, but not by strong acids. Notably, the degree of channel activation was predicted by the degree of intracellular acidification produced by each acid, suggesting that intracellular protons are the proximate stimulus that gates the channel. Responses to weak acids produced a Ca2+-independent inactivation that precluded further activation by weak acids or reactive chemicals, whereas preactivation by reactive electrophiles sensitized TRPA1 channels to weak acids. Importantly, responses of trigeminal neurons to weak acids were highly overrepresented in the subpopulation of TRPA1-expressing neurons and were severely reduced in neurons from TRPA1 knockout mice. We conclude that TRPA1 is a general sensor for weak acids that produce intracellular acidification and suggest that it functions within the pain pathway to mediate sensitivity to cellular acidosis.

Vagal Sensory Neuron Subtypes that Differentially Control Breathing

Breathing is essential for survival and under precise neural control. The vagus nerve is a major conduit between lung and brain required for normal respiration. Here, we identify two populations of mouse vagus nerve afferents (P2ry1, Npy2r), each a few hundred neurons, that exert powerful and opposing effects on breathing. Genetically guided anatomical mapping revealed that these neurons densely innervate the lung and send long-range projections to different brainstem targets. Npy2r neurons are largely slow-conducting C fibers, while P2ry1 neurons are largely fast-conducting A fibers that contact pulmonary endocrine cells (neuroepithelial bodies). Optogenetic stimulation of P2ry1 neurons acutely silences respiration, trapping animals in exhalation, while stimulating Npy2r neurons causes rapid, shallow breathing. Activating P2ry1 neurons did not impact heart rate or gastric pressure, other autonomic functions under vagal control. Thus, the vagus nerve contains intermingled sensory neurons constituting genetically definable labeled lines with different anatomical connections and physiological roles.

Piezo2 senses airway stretch and mediates lung inflation-induced apnoea

Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2+ vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired Hering–Breuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults.

The K+ channel KIR2.1 functions in tandem with proton influx to mediate sour taste transduction

Significance Among the five basic tastes, sour is one of the least understood. Notably, the sour receptor remains to be identified, and molecular mechanisms by which sour stimuli are detected are largely not known. Previous work has shown that H+ ions can directly enter sour taste cells, eliciting a change in membrane potential and acidification of the cell cytosol. In the present work, we identify a second element of sensory transduction, a K+ channel, KIR2.1, which is inhibited by intracellular acidification. The presence of an acid-sensitive K+ channel in sour taste cells allows for amplification of the sensory response and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids. Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.

Effects of insulin-like growth factor 1 on synaptic excitability in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory and neuroendocrine actions, and it also prevents beta amyloid-induced death of hippocampal neurons. However, its functions in the synaptic excitability remain uncertain. Here we investigated the effects of IGF-1 on synaptic excitability in cultured rat hippocampal neurons using whole-cell patch clamp recordings. Incubation the hippocampal neurons with different concentrations of IGF-1 for 24 h or 30 min significantly increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on the frequency of miniature EPSCs (mEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs). The mean amplitudes, rise, and decay kinetics of sEPSCs, mEPSCs, and sIPSCs were not significantly affected by IGF-1, indicating that IGF-1 increased the probability of neurotransmitter release but did not modulate postsynaptic receptors. The effects of IGF-1 were mediated by mitogen-activated protein kinase (MAPK). IGF-1 activated the ERK1/2 signaling pathway in cultured hippocampal neurons, and the inhibitor PD98059 blocked the enhancement of sEPSCs induced by IGF-1. These results demonstrated the regulatory function of IGF-1 on synaptic excitability in hippocampal neurons and its underlying signaling mechanism.

Effects of insulin‐like growth factor‐1 on okadaic acid‐induced apoptosis in SH‐SY5Y cells

The effects of insulin‐like growth factor‐1 (IGF‐1) on the cytotoxicity and apoptosis induced by okadaic acid (OA) in SH‐SY5Y cells were investigated. Cell viability was measured using the MTT (3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide) assay. Early and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide (PI) double‐staining. Caspase‐3 activation was detected by Western blot analysis. Preincubation with IGF‐1 for 24 h prevented cytotoxicity induced by 40 nM OA given for 24 h, and the MTT value significantly increased. Incubation with 20 nM OA for 24 h caused a marked increase in the percentage of early apoptotic and late apoptotic/necrotic cells, which was not dependent on the activation of caspase‐3. OA‐induced apoptosis was significantly decreased by pretreatment with 10 ng/ml of IGF‐1 for 24 h. The results supported the hypothesis that IGF‐1 may be useful in the treatment of Alzheimers disease.