Project description:Taste substances are received by taste receptors expressed in taste cells. “Salty taste” sensation is evoked when sodium and chloride ions are present together in the oral cavity. The presence of an epithelial cation channel that receives Na+ has previously been reported. However, no molecular entity involving Cl- receptors has been elucidated. We report the strong expression of transmembrane channel-like 4 (TMC4) in the circumvallate and foliate papillae projected to the glossopharyngeal nerve, mediating a high-concentration of NaCl. Electrophysiological analysis using HEK293T cells revealed that TMC4 was a voltage-dependent Cl- channel and the consequent currents were completely inhibited by NPPB, an anion channel blocker. This channel could be activated without an increase in intracellular calcium ion. TMC4 allowed permeation of organic anions including gluconate, but their current amplitudes at positive potentials were less than that of Cl-. Tmc4-deficient mice showed significantly weaker glossopharyngeal nerve response to high-concentration of NaCl than the wild-type littermates. These results indicated that TMC4 is a novel chloride channel that responds to high-concentration of NaCl.
Project description:The sense of taste is an important sentinel governing what should or should not be ingested by an animal, with high pH sensation playing a critical role in food selection. Here we explore the molecular identities of taste receptors detecting the basic pH of food using Drosophila melanogaster as a model. We identify a chloride channel named alkaliphile (Alka), which is both necessary and sufficient for aversive taste responses to basic food. Alka forms a high-pH-gated chloride channel and is specifically expressed in a subset of gustatory receptor neurons (GRNs). Optogenetic activation of alka-expressing GRNs is sufficient to suppress attractive feeding responses to sucrose. Conversely, inactivation of these GRNs causes severe impairments in the aversion to high pH. Altogether, our discovery of Alka as an alkaline taste receptor lays the groundwork for future research on alkaline taste sensation in other animals.
Project description:The proton-activated chloride (PAC) channel (also known as acid-sensitive outwardly rectifying anion channel, ASOR) plays a critical role in acid-induced cell death and endocytosis. However, little is known about the regulatory factors and binding partners of PAC. In this study, we discovered that transfer RNA (tRNA) interacts directly with PAC as an unexpected binding partner. Using cryo-electron microscopy, we determined that two PAC trimers and one co-purified tRNA molecule form a stable complex via a highly conserved KR motif, representing the closed conformation. tRNA is located on the intracellular side of PAC, blocking the channel pores. Furthermore, electrophysiological data showed that tRNA modulates chloride currents and channel open probability of PAC, thus protecting against acid-induced cell death. Our study provides insight into the regulation of PAC activity by cytosolic tRNA and extends the role of tRNAs in pathological and physiological events.
Project description:Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. The first comprehensive database of gene expression in primate (Macaca fascicularis) taste buds is presented. This database provides a foundation for further studies in diverse aspects of taste biology. A taste bud gene expression database was generated using laser capture microdissection (LCM) of tissue freeze medium OTC embedded macaque tongue tissue blocks. We collected fungiform (FG) taste buds at the front of the tongue, circumvallate (CV) taste buds at the back of the tongue, as well as non-gustatory lingual epithelium (LE). Gene expression was also analyzed in the top and bottom portions of CV taste buds collected using LCM. Samples were collected from 10 animals - 7 female, 3 male.
Project description:Methylene diphenyl diisocyanate is a chemical known to cause asthma. The present study uses mice to investigate exposure-induced changes in lung gene expression and effects of a chloride channel inhibitor We used microarrays to detail global whole lung gene expression following respiratory tract exposure to methylene diphenyl diisocyanate (MDI) vs. control exposure in mice immune-sensitized to MDI by prior skin exposure. In some studies mice were given a chloride channel inhibitor (crofelemer) via the respiratory tract before MDI.
Project description:Primary aldosteronism (PA), a common cause of severe hypertension, features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) and 80 additional probands with unsolved early-onset PA. Eight probands had novel heterozygous variants in CLCN2, including two de novo mutations and four independent occurrences of the identical p.Arg172Gln mutation; all relatives with early-onset PA carried the CLCN2 variant found in probands. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. In this data set, we examined RNA expression in H295R cells transfected with empty vector, WT and p.Arg172Gln CLCN2. Expression of CLCN2 led to increased expression of CYP11B2 and its upstream regulator NR4A2.
Project description:Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclasts remains unresolved. Here, we identify the existence of Anoctamin 1 in osteoclasts and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl- concentration, decreases H+ secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis.
Project description:Numerous studies show dietary carbohydrates (C) affect the sensation of sweetness. However, protein (P) is one of the most critical macronutrients in the diet as well. It is still unclear how carbohydrates and proteins interact to influence sweet taste sensitivity. Here, we use the nutritional geometry framework (NGF) to tackle this problem in Drosophila melanogaster. Our results showed that the combination of high protein and low carbohydrates caused higher taste responses to sucrose stimuli in both sexes. Additionally, transcriptome analysis revealed that the gene expression of glycine, serine, and threonine pathway in the high-protein, low-carbohydrate diet was significantly upregulated, compared to a diet with low protein, and high carbohydrate. We confirmed that serine and threonine supplementation in the high-carbohydrate, low-protein diet enhanced the sucrose sensitivity of flies. Our results demonstrate that sucrose taste sensitivity is affected by the dietary balance of protein and carbohydrates possibly mediated by the change in serine, and threonine. The high protein, low carbohydrate diets enhanced sucrose taste sensitivity.
Project description:The ability to sense sour provides an important sensory signal to prevent the ingestion of unripe, spoiled or fermented foods. Taste and somatosensory receptors in the oral cavity trigger aversive behaviors in response to acid stimuli. Here we show that the ion channel Otopetrin-1, a proton-selective channel normally involved in the sensation of gravity in the vestibular system, is essential for sour-sensing in the taste system. We demonstrate that a knockout of Otop1 eliminates acid responses from sour-sensing taste-receptor-cells (TRCs). In addition, we show that mice engineered to express otopetrin-1 in sweet TRCs now have sweet cells that also respond to sour stimuli. Next, we genetically identified the taste ganglion neurons mediating each of the five basic taste qualities, and demonstrate that sour taste uses its own dedicated labeled line from TRCs in the tongue to finely tuned taste neurons in the brain to trigger aversive behaviors.