TRPM4 and TRPM5 are both required for normal signaling in taste receptor cells.
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ABSTRACT:
SUBMITTER: Dutta Banik D
PROVIDER: S-EPMC5789955 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nicotine evokes chorda tympani (CT) taste nerve responses and an aversive behavior in Trpm5 knockout (KO) mice. The agonists and antagonists of nicotinic acetylcholine receptors (nAChRs) modulate neural and behavioral responses to nicotine in wildtype (WT) mice, Trpm5 KO mice and rats. This indicates that nicotine evokes bitter taste by activating a Trpm5-dependent pathway and a Trpm5-independent but nAChR-dependent pathway. Rat CT responses to ethanol are also partially inhibited by nAChR blockers, mecamylamine and dihydro-β-erythroidine. This indicates that a component of the bitter taste of ethanol is also nAChR-dependent. However, at present the expression and localization of nAChR subunits has not been investigated in detail in taste receptor cells (TRCs). To this end, in situ hybridization, immunohistochemistry and q-RT-PCR techniques were utilized to localize nAChR subunits in fungiform and circumvallate TRCs in WT mice, Trpm5-GFP transgenic mice, nAChR KO mice, and rats. The expression of mRNAs for α7, β2 and β4 nAChR subunits was observed in a subset of rat and WT mouse circumvallate and fungiform TRCs. Specific α3, α4, α7, β2, and β4 antibodies localized to a subset of WT mouse circumvallate and fungiform TRCs. In Trpm5-GFP mice α3, α4, α7, and β4 antibody binding was observed in a subset of Trpm5-positive circumvallate TRCs. Giving nicotine (100 μg/ml) in drinking water to WT mice for 3 weeks differentially increased the expression of α3, α4, α5, α6, α7, β2 and β4 mRNAs in circumvallate TRCs to varying degrees. Giving ethanol (5%) in drinking water to WT mice induced an increase in the expression of α5 and β4 mRNAs in circumvallate TRCs with a significant decrease in the expression of α3, α6 and β2 mRNAs. We conclude that nAChR subunits are expressed in Trpm5-positive TRCs and their expression levels are differentially altered by chronic oral exposure to nicotine and ethanol.
| S-EPMC5749851 | biostudies-literature
Project description:BACKGROUND AND PURPOSE: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. EXPERIMENTAL APPROACH: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. KEY RESULTS: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. CONCLUSIONS AND IMPLICATIONS: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.
| S-EPMC2438271 | biostudies-other
Project description:The transient receptor potential channel, TRPM4, and its closest homolog, TRPM5, are non-selective cation channels that are activated by an increase in intracellular calcium. They are expressed in many cell types, including neurons and myocytes. Although the electrophysiological and pharmacological properties of these two channels have been previously studied, less is known about their regulation, in particular their post-translational modifications. We, and others, have reported that wild-type (WT) TRPM4 channels expressed in HEK293 cells, migrated on SDS-PAGE gel as doublets, similar to other ion channels and membrane proteins. In the present study, we provide evidence that TRPM4 and TRPM5 are each N-linked glycosylated at a unique residue, Asn(992) and Asn(932), respectively. N-linked glycosylated TRPM4 is also found in native cardiac cells. Biochemical experiments using HEK293 cells over-expressing WT TRPM4/5 or N992Q/N932Q mutants demonstrated that the abolishment of N-linked glycosylation did not alter the number of channels at the plasma membrane. In parallel, electrophysiological experiments demonstrated a decrease in the current density of both mutant channels, as compared to their respective controls, either due to the Asn to Gln mutations themselves or abolition of glycosylation. To discriminate between these possibilities, HEK293 cells expressing TRPM4 WT were treated with tunicamycin, an inhibitor of glycosylation. In contrast to N-glycosylation signal abolishment by mutagenesis, tunicamycin treatment led to an increase in the TRPM4-mediated current. Altogether, these results demonstrate that TRPM4 and TRPM5 are both N-linked glycosylated at a unique site and also suggest that TRPM4/5 glycosylation seems not to be involved in channel trafficking, but mainly in their functional regulation.
| S-EPMC3932411 | biostudies-literature
Project description:Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by ? cells.
| S-EPMC4665783 | biostudies-literature
Project description:The orosensory responses elicited by nicotine are relevant for the development and maintenance of addiction to tobacco products. However, although nicotine is described as bitter tasting, the molecular and neural substrates encoding the taste of nicotine are unclear. Here, rats and mice were used to determine whether nicotine activates peripheral and central taste pathways via TRPM5-dependent mechanisms, which are essential for responses to other bitter tastants such as quinine, and/or via nicotinic acetylcholine receptors (nAChRs). When compared with wild-type mice, Trpm5(-/-) mice had reduced, but not abolished, chorda tympani (CT) responses to nicotine. In both genotypes, lingual application of mecamylamine, a nAChR-antagonist, inhibited CT nerve responses to nicotine and reduced behavioral responses of aversion to this stimulus. In accordance with these findings, rats were shown to discriminate between nicotine and quinine presented at intensity-paired concentrations. Moreover, rat gustatory cortex (GC) neural ensemble activity could also discriminate between these two bitter tastants. Mecamylamine reduced both behavioral and GC neural discrimination between nicotine and quinine. In summary, nicotine elicits taste responses through peripheral TRPM5-dependent pathways, common to other bitter tastants, and nAChR-dependent and TRPM5-independent pathways, thus creating a unique sensory representation that contributes to the sensory experience of tobacco products.
| S-EPMC2635785 | biostudies-literature
Project description:Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-?B in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca2+-dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.
| S-EPMC5667964 | biostudies-literature
Project description:Knockout (KO) mice missing the sweet taste receptor subunit T1R3 or the signaling protein TRPM5 have greatly attenuated sweetener preferences. Yet both types of KO mice develop preferences for glucose but not fructose in 24-h tests, which has been attributed to the postoral reinforcing actions of glucose. Here we probed for residual sugar taste sensitivity in KO mice. Unlike wildtype (WT) mice, food-restricted T1R3 KO and TRPM5 KO mice displayed little attraction for 8% glucose and 8% fructose in 1-min, two-bottle choice tests. However, in 1-h tests about half of the T1R3 KO mice displayed a significant preference for glucose over fructose (78-84%), while WT mice showed either no or weak preferences (41-56%) for glucose. Following one-bottle training sessions, WT mice display greater glucose preferences although still weaker than those observed in T1R3 KO mice. In contrast, TRPM5 KO mice were indifferent to sugars in 1-h tests but developed a strong preference for glucose over fructose in 24-h tests. T1R3 taste cells contain the sodium glucose cotransporter 1 (SGLT1) and the ATP-gated K+ (KATP) metabolic sensor, which may mediate the unlearned glucose preference displayed by T1R3 KO mice. Unlike WT mice, many T1R3 KO mice strongly preferred glucose to a non-metabolizable glucose analog (α-methyl-D-glucopyranoside, MDG) in initial 1-h choice tests. Glucose and MDG are both ligands for SGLT1 which indicates that SGLT1 sensing does not mediate the glucose preference of T1R3 KO mice. Instead, KATP sensing and/or other oral sensors are implicated. The MDG findings also argue against postoral sensing as the primary source of the initial glucose preference displayed by T1R3 KO mice. Why only half of the T1R3 KO mice showed this preference in 1-h tests remains to be determined. All T1R3 KO mice preferred glucose to fructose in 24-h tests, which appears to be due to both oral and postoral glucose sensing.
| S-EPMC7417196 | biostudies-literature
Project description:Gustatory neurons transmit chemical information from taste receptor cells, which reside in taste buds in the oral cavity, to the brain. As adult taste receptor cells are renewed at a constant rate, nerve fibers must reconnect with new taste receptor cells as they arise. Therefore, the maintenance of gustatory innervation to the taste bud is an active process. Understanding how this process is regulated is a fundamental concern of gustatory system biology. We speculated that because brain-derived neurotrophic factor (BDNF) is required for taste bud innervation during development, it might function to maintain innervation during adulthood. If so, taste buds should lose innervation when Bdnf is deleted in adult mice. To test this idea, we first removed Bdnf from all cells in adulthood using transgenic mice with inducible CreERT2 under the control of the Ubiquitin promoter. When Bdnf was removed, approximately one-half of the innervation to taste buds was lost, and taste buds became smaller because of the loss of taste bud cells. Individual taste buds varied in the amount of innervation each lost, and those that lost the most innervation also lost the most taste bud cells. We then tested the idea that that the taste bud was the source of this BDNF by reducing Bdnf levels specifically in the lingual epithelium and taste buds. Taste buds were confirmed as the source of BDNF regulating innervation. We conclude that BDNF expressed in taste receptor cells is required to maintain normal levels of innervation in adulthood.
| S-EPMC4697083 | biostudies-literature
Project description:Experimental or traumatic nerve injury causes the degeneration of associated taste buds. Unlike most sensory systems, the sectioned nerve and associated taste buds can then regenerate, restoring neural responses to tastants. It was previously unknown whether injury-induced immune factors mediate this process. The proinflammatory cytokines, interleukin (IL)-1α and IL-1β, and their requisite receptor are strongly expressed by anterior taste buds innervated by the chorda tympani nerve. We tested taste bud regeneration and functional recovery in mice lacking the IL-1 receptor. After axotomy, the chorda tympani nerve regenerated but was initially unresponsive to tastants in both WT and Il1r KO mice. In the absence of Il1r signaling, however, neural taste responses remained minimal even >8 weeks after injury in both male and female mice, whereas normal taste function recovered by 3 weeks in WT mice. Failed recovery was because of a 57.8% decrease in regenerated taste buds in Il1r KO compared with WT axotomized mice. Il1a gene expression was chronically dysregulated, and the subset of regenerated taste buds were reinnervated more slowly and never reached full volume as progenitor cell proliferation lagged in KO mice. Il1r signaling is thus required for complete taste bud regeneration and the recovery of normal taste transmission, likely by impairing taste progenitor cell proliferation. This is the first identification of a cytokine response that promotes taste recovery. The remarkable plasticity of the taste system makes it ideal for identifying injury-induced mechanisms mediating successful regeneration and recovery.SIGNIFICANCE STATEMENT Taste plays a critical role in nutrition and quality of life. The adult taste system is highly plastic and able to regenerate following the disappearance of most taste buds after experimental nerve injury. Several growth factors needed for taste bud regeneration have been identified, but we demonstrate the first cytokine pathway required for the recovery of taste function. In the absence of IL-1 cytokine signaling, taste bud regeneration is incomplete, preventing the transmission of taste activity to the brain. These results open a new direction in revealing injury-specific mechanisms that could be harnessed to promote the recovery of taste perception after trauma or disease.
| S-EPMC10184746 | biostudies-literature
Project description:Solitary chemosensory cells (SCCs) are specialized cells in the respiratory epithelium that respond to noxious chemicals including bacterial signaling molecules. SCCs express components of bitter taste transduction including the taste receptor type 2 (TAS2R) bitter taste receptors and downstream signaling effectors: ?-Gustducin, phospholipase C?2 (PLC?2), and transient receptor potential cation channel subfamily M member 5 (TRPM5). When activated, SCCs evoke neurogenic reflexes, resulting in local inflammation. The purpose of this study was to test for the presence SCCs in human sinonasal epithelium, and to test for a correlation with inflammatory disease processes such as allergic rhinitis and chronic rhinosinusitis.Patient demographics and biopsies of human sinonasal mucosa were obtained from control patients (n = 7) and those with allergic rhinitis and/or chronic rhinosinusitis (n = 15). Reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), and immunohistochemistry were used to determine whether expression of signaling effectors was altered in diseased patients.RT-PCR demonstrated that bitter taste receptors TAS2R4, TAS2R14, and TAS2R46, and downstream signaling effectors ?-Gustducin, PLC?2, and TRPM5 are expressed in the inferior turbinate, middle turbinate, septum, and uncinate of both control and diseased patients. PLC?2/TRPM5-immunoreactive SCCs were identified in the sinonasal mucosa of both control and diseased patients. qPCR showed similar expression of ?-Gustducin and TRPM5 in the uncinate process of control and diseased groups, and there was no correlation between level of expression and 22-item Sino-Nasal Outcomes Test (SNOT-22) or pain scores.SCCs are present in human sinonasal mucosa in functionally relevant areas. Expression level of signaling effectors was similar in control and diseased patients and did not correlate with measures of pain and inflammation. Further study into these pathways may provide insight into nasal inflammatory diseases and may offer potential therapeutic targets.
| S-EPMC3655139 | biostudies-literature