Bachelor of Science, Zhejiang University (2000)
Doctor of Philosophy, Hong Kong University Of Science & Technology (2009)
Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.
View details for DOI 10.1016/j.neuron.2014.01.044
View details for Web of Science ID 000333326000012
This chapter describes four different protocols used to assay thermotaxis navigation behavior of single, or populations of, C. elegans hermaphrodites on spatial thermal gradients within the physiological temperature range (15-25°C). A method to assay avoidance of noxious temperatures is also described.
View details for DOI 10.1895/wormbook.1.168.1
View details for PubMedID 24563245
Certain thermoreceptor neurons are sensitive to tiny thermal fluctuations (0.01°C or less) and maintain their sensitivity across a wide range of ambient temperatures through a process of adaptation, but understanding of the biochemical basis for this performance is rudimentary. Prior studies of the AFD thermoreceptor in Caenorhabditis elegans revealed a signaling cascade that depends on a trio of receptor guanylate cyclases (rGCs), GCY-8, GCY-18, and GCY-23, and gives rise to warming-activated thermoreceptor currents (ThRCs) carried by cyclic GMP-gated ion channels. The threshold for ThRC activation adapts to the ambient temperature through an unknown calcium-dependent process. Here, we use in vivo whole-cell patch-clamp recording from AFD to show that loss of GCY-8, but not of GCY-18 or GCY-23, reduces or eliminates ThRCs, identifying this rGC as a crucial signaling element. To learn more about thermotransduction and adaptation, we used behavioral screens and analysis of gene expression patterns to identify phosphodiesterases (PDEs) likely to contribute to thermotransduction. Deleting PDE-2 decouples the threshold for ThRC activation from ambient temperature, altering adaptation. We provide evidence that the conserved neuronal calcium sensor 1 protein also regulates the threshold for ThRC activation and propose a signaling network to account for ThRC activation and adaptation. Because PDEs play essential roles in diverse biological processes, including vertebrate phototransduction and olfaction, and regulation of smooth muscle contractility and cardiovascular function, this study has broad implications for understanding how extraordinary sensitivity and dynamic range is achieved in cyclic nucleotide-based signaling networks.
View details for DOI 10.1085/jgp.201310959
View details for Web of Science ID 000325278400010
View details for PubMedID 24081984
CFTR function is tightly regulated by many interacting proteins.Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR.K18 controls the function of CFTR.These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.
View details for DOI 10.1074/jbc.M112.403584
View details for Web of Science ID 000311448800045
View details for PubMedID 23045527
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion and intracellular ligand-gated channel associated with cystic fibrosis, a lethal genetic disorder common among Caucasians. Here we show that CFTR is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular and tissue levels. Stretch increased the product of the number of channels present and probability of being open (NPo), and also increased the unitary conductance of CFTR in cell-attached membrane patches. CFTR stretch-mediated activation appears to be an intrinsic property independent of cytosolic factors and kinase signalling. CFTR stretch-mediated activation resulted in chloride transport in Calu-3 human airway epithelial cells and mouse intestinal tissues. Our study has revealed an unexpected function of CFTR in mechanosensing, in addition to its roles as a ligand-gated anion channel and a regulator of other membrane transporters, demonstrating for the first time a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation in vivo.
View details for DOI 10.1038/ncb2053
View details for Web of Science ID 000277283700017
View details for PubMedID 20400957
Cystic fibrosis transmembrane conductance regulator (CFTR) has been found to be colocalized with G-protein-coupled receptors (GPCRs) and the downstream signaling molecules; however, the mechanisms of the colocalization remain largely elusive. The present work has investigated the role of lipid rafts in the localized signaling from GPCRs to CFTR. Using commonly used sucrose gradient centrifugation, we found that CFTR along with G(alpha)S was associated with lipid rafts, and the association was disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MCD) treatment in Calu-3 human airway epithelial cells. Using short-circuit current (I (sc)) as a readout of CFTR in Calu-3 cells or T84 human colonic epithelial cells, we showed that MCD, while increasing basal membrane permeability, had no effect on the I (sc) induced by several GPCR agonists. Similar results were also obtained with a cholesterol biosynthesis inhibitor lovastatin and a cholesterol-binding agent filipin in Calu-3 cells. Furthermore, cholesterol depletion did not impair cyclic AMP production elicited by the GPCR agonists in Calu-3 cells. Our data suggest that GPCR-mediated signaling maintain their integrity after lipid raft disruption in Calu-3 and T84 epithelial cells and cast doubts on the role of lipid rafts as signaling platforms in GPCR-mediated signaling.
View details for DOI 10.1007/s00424-008-0460-2
View details for Web of Science ID 000256825000013
View details for PubMedID 18224335
In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current (Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.
View details for DOI 10.1152/ajpcell.00556.2007
View details for Web of Science ID 000256574900019
View details for PubMedID 18385283