The odorant receptor OR2W3 on airway smooth muscle evokes bronchodilation via a cooperative chemosensory tradeoff between TMEM16A and CFTR.

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca2+]i) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca2+]i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca2+]i mobilization and ASM relaxation required Ca2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca2+-activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases

Identification and characterization of novel renal sensory receptors.

Recent studies have highlighted the important roles that "sensory" receptors (olfactory receptors, taste receptors, and orphan "GPR" receptors) play in a variety of tissues, including the kidney. Although several studies have identified important roles that individual sensory receptors play in the kidney, there has not been a systematic analysis of the renal repertoire of sensory receptors. In this study, we identify novel renal sensory receptors belonging to the GPR (n = 76), olfactory receptor (n = 6), and taste receptor (n = 11) gene families. A variety of reverse transcriptase (RT)-PCR screening strategies were used to identify novel renal sensory receptors, which were subsequently confirmed using gene-specific primers. The tissue-specific distribution of these receptors was determined, and the novel renal ORs were cloned from whole mouse kidney. Renal ORs that trafficked properly in vitro were screened for potential ligands using a dual-luciferase ligand screen, and novel ligands were identified for Olfr691. These studies demonstrate that multiple sensory receptors are expressed in the kidney beyond those previously identified. These results greatly expand the known repertoire of renal sensory receptors. Importantly, the mRNA of many of the receptors identified in this study are expressed highly in the kidney (comparable to well-known and extensively studied renal GPCRs), and in future studies it will be important to elucidate the roles that these novel renal receptors play in renal physiology

Summary of the tissue expression profile of all the novel sensory receptors identified in the mouse whole kidney cDNA.

<p>A ‘+’ sign indicates expression of the corresponding receptor in our RT-PCR screen whereas a ‘−’ sign indicates absence in that particular tissue. All ‘+’ signs in the table were confirmed by sequencing to confirm identity. In each case, the mock sample without reverse transcriptase during cDNA synthesis was negative.</p><p>Summary of the tissue expression profile of all the novel sensory receptors identified in the mouse whole kidney cDNA.</p

RT-PCR with mouse whole kidney cDNA as template to identify novel renal olfactory receptors.

<p>Olfr99 (A), Olfr545 (B), Olfr691 (C), Olfr693 (D), Olfr31(E) and Olfr1426(F) expression is detectible in mouse whole kidney cDNA by PCR and sequencing confirms the identity of amplified products. Mock RT template controls are negative for OR GSP sets and β-actin (not shown). The white arrow indicates the band of the expected size for each olfactory receptor.</p

RT-PCR with mouse whole kidney cDNA as template to identify novel renal taste receptors.

<p>Tas2r108 (A), Tas2r119 (B), Tas2r135 (C), Tas2r137 (D), Tas2r138 (E), Tas2r140 (F), Tas2r143 (G), Tas1r1 (H), Tas1r2 (I) and Tas1r3 (J) PKD1L3 (K) and G_NAT3 (L) expression detected in the mouse whole kidney cDNA by RT-PCR and confirmed by sequencing. Mock controls without RT are negative in all the lanes. The white arrow indicates the band of the expected size for each olfactory receptor.</p

Dose response curves for the novel Olfr691 ligands.

<p>Dose response curves show that Olfr691 has the highest affinity for valproate when co-expressed with RTP1S in HEK293T cells, with an EC₅₀ value of 0.4778 mM; however 4-pentenoate induced the strongest cAMP responses at all doses when compared to isobutyrate, valerate and valproate. NT represents measurements obtained from non-treated cells (with no ligand) transfected with Olfr691 and RTP1S.</p

Immunohistochemistry showing surface expression of ORs.

<p>Each OR is shown under the experimentally determined condition which allowed for optimized surface expression in HEK293T cells. The surface trafficking conditions vary for each OR and we have published the corresponding conditions for Olfr99, 545, 691 and 693 previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone.0111053-Shepard1" target="_blank">[25]</a>. Briefly, Olfr31 requires co-expression of RTP1S; Olfr691 & Olfr693 require presence of N-terminal Lucy tag along with co-expression of RTP1S; Olfr99, Olfr545 & Olfr1426 requires presence of N-terminal Lucy tag along with co-expression of RTP1S and Ric8b respectively. Olfr31 requires co-expression of RTP1S and Olfr1426 failed to reach the membrane surface at all the tested conditions. HEK293T cells were first stained with a poly-flag antibody (surface) then subsequently permeabilized and stained with a mono-flag antibody (total). The images were taken at equal exposure between all surface and total conditions. Surface images are marked with either a+or – in their lower right-hand corners to indicate the presence or absence of surface expression, respectively. Images in (B) have been enhanced to better display surface expression. Images in (A) are presented as they were taken. Unenhanced images for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone-0111053-g004" target="_blank">Figure 4B</a> can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111053#pone.0111053.s001" target="_blank">Figure S1</a>.</p

Ligand screening for Olfr691.

<p>Olfr691 responds to published short chain fatty acids, isovalerate and valerate, in a dose dependent manner when co-expressed with RTP1S (A). Further ligand screening shows that Olfr691 responds to wide range of saturated short and medium chained fatty acids, from propionate to octanoate, but not including formate and acetate (B). NT represents measurements obtained from non-treated cells (with no ligand) transfected with Olfr691 and RTP1S.</p