Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

919

966

Olfactory receptors are G protein-coupled receptors that mediate olfactory chemosensation and serve as chemosensors in other tissues. We find that Olfr78, an olfactory receptor expressed in the kidney, responds to short chain fatty acids (SCFAs). Olfr78 is expressed in the renal juxtaglomerular apparatus, where it mediates renin secretion in response to SCFAs. In addition, both Olfr78 and G protein-coupled receptor 41 (Gpr41), another SCFA receptor, are expressed in smooth muscle cells of small resistance vessels. Propionate, a SCFA shown to induce vasodilation ex vivo, produces an acute hypotensive response in wild-type mice. This effect is differentially modulated by disruption of Olfr78 and Gpr41 expression. SCFAs are end products of fermentation by the gut microbiota and are absorbed into the circulation. Antibiotic treatment reduces the biomass of the gut microbiota and elevates blood pressure in Olfr78 knockout mice. We conclude that SCFAs produced by the gut microbiota modulate blood pressure via Olfr78 and Gpr41.

Section: Resultsmentioning

confidence: 99%

“…Surface immunofluorescence (nonpermeablized stain) and surface ELISA were performed as previously described (24,48). Wells for surface ELISA were assayed in quadruplicate.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation

Pluznick

Protzko

Gevorgyan

et al. 2013

919

966

“…Hana3A cells, a modified HEK293 cell line, stably express RTP1L (receptor-transporting protein 1L), RTP2, REEP1 (receptor expression-enhancing protein 1), and G αolf to facilitate OR surface expression (44). Hana3A cells were grown in MEM (Cellgro), supplemented with 10% (vol/vol) FBS (Cellgro), 100 μg/mL penicillin-steptomycin (Invitrogen), 1.25 g/mL amphotericin (Sigma), and 1 μg/mL puromycin (Sigma).…”

Section: Methodsmentioning

confidence: 99%

G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

Connelly

Grosmaître

et al. 2014

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR-G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction. , but our understanding of the mechanical sensors is still limited. We previously discovered that some OSNs in the mammalian nose responded to mechanical stimulation (4), a feature that may allow the nose to carry an afferent signal of breathing to the brain and facilitate binding of orofacial sensation (5). In the current study, we aim to identify the mechanical sensor(s) and mechanotransduction pathway in OSNs.In mammals, smell perception depends on a large family of ORs expressed in OSNs. Out of a repertoire of >1,000 ORs (6, 7), each OSN expresses a single type, which determines its response profile and central target in the brain. Binding of odorant molecules with specific ORs activates the olfactory G protein G olf , which in turn activates type III adenylyl cyclase (ACIII). ACIII activation causes increased production of cAMP, which opens a cyclic nucleotide-gated cation (CNG) channel. The inward current via the CNG channel is further amplified by Cl − outflow through a calcium-activated Cl − channel. This transduction cascade leads to depolarization of OSNs, which fire action potentials carrying the odor information to the brain (8). OSNs expressing the same OR are scattered in one of the few broadly defined zones in the olfactory epithelium, but their axons typically converge onto a pair of glomeruli in the olfactory bulb (9).Here we report that disruption of the olfactory signal transduction cascade completely eliminates mechanical responses in OSNs. OSNs expressing different receptor types display differential responses to mechanical stimuli. For instance, I7, M71, and SR1 neurons have much stronger mechanical responses than MOR23 and mOR-EG neurons. Loss-of-function mutation of the I7 receptor, genetic switch of the M71 receptor, or ablation of the SR1 receptor, abolishes or dramatically reduces mechanical responses in the host OSNs. Furthermore, ectopic expression of the I7 receptor restores mechanosensitivity in loss-of-function mutant I7 cells. ...

“…The Dual-Glo Luciferase Assay (Promega) was used to determine the activities of firefly and Renilla luciferase in Hana3A cells (26). Firefly luciferase, driven by a cAMP response element promoter (CRE-Luc; Stratagene), was used to determine OR activation levels.…”

Section: Methodsmentioning

confidence: 99%

Responsiveness of G protein-coupled odorant receptors is partially attributed to the activation mechanism

March²,

et al. 2015

Self Cite

Mammals detect and discriminate numerous odors via a large family of G protein-coupled odorant receptors (ORs). However, little is known about the molecular and structural basis underlying OR response properties. Using site-directed mutagenesis and computational modeling, we studied ORs sharing high sequence homology but with different response properties. When tested in heterologous cells by diverse odorants, MOR256-3 responded broadly to many odorants, whereas MOR256-8 responded weakly to a few odorants. Out of 36 mutant MOR256-3 ORs, the majority altered the responses to different odorants in a similar manner and the overall response of an OR was positively correlated with its basal activity, an indication of ligand-independent receptor activation. Strikingly, a single mutation in MOR256-8 was sufficient to confer both high basal activity and broad responsiveness to this receptor. These results suggest that broad responsiveness of an OR is at least partially attributed to its activation likelihood.