Wallrabenstein I et al. (2013), Human trace amine-associated receptor TAAR5 can...

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PLoS One 2013 Jan 01;82:e54950. doi: 10.1371/journal.pone.0054950.

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Human trace amine-associated receptor TAAR5 can be activated by trimethylamine.

Wallrabenstein I , Kuklan J , Weber L , Zborala S , Werner M , Altmüller J , Becker C , Schmidt A , Hatt H , Hummel T , Gisselmann G .

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In addition to the canonical olfactory receptors, TAARs were currently suggested to be a second class of chemosensory receptors in the olfactory epithelium of vertebrates. In contrast to several deorphanized murine TAARs, agonists for the intact human TAAR genes 2, 5, 6, 8 and 9 that are potentially expressed in the human olfactory epithelium have not been determined so far. Moreover, the physiological relevance of TAARs still remains elusive. We present the first successful functional expression of a human TAAR and agonists of human TAAR5. We performed a ligand screening using recombinantly expressed human TAAR5 in HANA3A cells and Xenopus laevis oocytes. In order to measure receptor activity, we used a cAMP-dependent reporter gene assay and two-electrode voltage clamp technique. As a result, human TAAR5 can be activated in a concentration-dependent manner by trimethylamine and with less efficacy by dimethylethylamine. It could neither be activated by any other of the tested single amines with a related chemical structure (42 in total), nor by any of the tested odorant mixtures. The hypothesis that Single Nucleotide Polymorphisms (SNP) within the reading frame of an olfactory receptor gene can cause a specific anosmia, formed the basis for clarifying the question, if anosmia for trimethylamine is caused by a SNP in a TAAR coding sequence. All functional human TAAR gene reading frames of subjects with specific anosmia for trimethylamine were amplified and products analyzed regarding SNP distribution. We demonstrated that the observed specific anosmia for trimethylamine is not correlated with a SNP in the coding sequence of one of the putatively functional human TAAR genes.

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Species referenced: Xenopus laevis
Genes referenced: camp cftr rho

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	Figure 2. Chemical structure of various tested TMA analogs.Only tertiary amines (1) trimethylamine and (2) dimethylethylamine can activate hTAAR5. (3) triethylamine, (4) diethylmethylamine, (5) dimethylamine, (6) methylamine, (7) trimethylphosphine, (8) cyclohexylamine, (9) N-methylpiperidine, (10) pyridine, (11) β-phenylethylamine, (12) skatole, (13) ethanolamine, (14) putrescine, (15) isobutylamine, (16) dimethylbutylamine.
	Figure 3. Human TAAR5 is selectively activated by TMA and DMEA.Responses of hTAAR5 to 42 different amines or amine-like substances. The concentration of all tested substances was 100 µM. Responses were normalized to the response to forskolin (10 µM). Data are given as mean ± SEM of 2–10 independent experiments, each performed in duplicates. TMA and DMEA induced signals significantly differing from mock-transfected controls (*p<0.001; p<0.01).
	Figure 4. TMA and DMEA act as agonists at the hTAAR5 receptor in a concentration-dependent manner.Human TAAR5 responses were normalized to the response to forskolin (10 µM). EC50 = 116 µM (TMA) and EC50 = 169 µM (DMEA). Detection threshold for TMA is 1 µM (p<0.05) and for DMEA 30 µM (p<0.05). Error bars represent SEM.
	Figure 5. TMA and DMEA activate hTAAR5 expressed in Xenopus oocytes. A:IBMX induced currents in oocytes expressing the reporter channel CFTR only. B: In oocytes injected with hTAAR5 cRNA application of TMA and DMEA as well as IBMX as positive control generated currents. C: Test substance induced currents normalized to the corresponding IBMX response. TMA and DMEA induced currents significantly differing from Ringer control (*p<0.001; p<0.01). Evoked currents were measured by two-electrode voltage clamp technique at a holding potential of −70 mM. Black bars indicate time of agonist application. Error bars represent SEM.
	Figure 1. Detection of the hTAAR5 receptor protein.Expression of the rhodopsin-tagged hTAAR5 receptor in transfected, fixed HANA3A cells was detected by the anti-rhodopsin antibody 4D2 and a secondary antibody labeled with the fluorescent dye Alexa Fluor 488 (green). Cell nuclei were stained by DAPI (blue). Left: Cells transfected with hTAAR5, right: mock-transfected control cells. Scaling bar: 20 µm.

References [+] :

Abaffy, Functional analysis of a mammalian odorant receptor subfamily. 2006, Pubmed, Xenbase

	Figure 2. Chemical structure of various tested TMA analogs.Only tertiary amines (1) trimethylamine and (2) dimethylethylamine can activate hTAAR5. (3) triethylamine, (4) diethylmethylamine, (5) dimethylamine, (6) methylamine, (7) trimethylphosphine, (8) cyclohexylamine, (9) N-methylpiperidine, (10) pyridine, (11) β-phenylethylamine, (12) skatole, (13) ethanolamine, (14) putrescine, (15) isobutylamine, (16) dimethylbutylamine.
	Figure 3. Human TAAR5 is selectively activated by TMA and DMEA.Responses of hTAAR5 to 42 different amines or amine-like substances. The concentration of all tested substances was 100 µM. Responses were normalized to the response to forskolin (10 µM). Data are given as mean ± SEM of 2–10 independent experiments, each performed in duplicates. TMA and DMEA induced signals significantly differing from mock-transfected controls (*p<0.001; p<0.01).
	Figure 4. TMA and DMEA act as agonists at the hTAAR5 receptor in a concentration-dependent manner.Human TAAR5 responses were normalized to the response to forskolin (10 µM). EC50 = 116 µM (TMA) and EC50 = 169 µM (DMEA). Detection threshold for TMA is 1 µM (p<0.05) and for DMEA 30 µM (p<0.05). Error bars represent SEM.
	Figure 5. TMA and DMEA activate hTAAR5 expressed in Xenopus oocytes. A:IBMX induced currents in oocytes expressing the reporter channel CFTR only. B: In oocytes injected with hTAAR5 cRNA application of TMA and DMEA as well as IBMX as positive control generated currents. C: Test substance induced currents normalized to the corresponding IBMX response. TMA and DMEA induced currents significantly differing from Ringer control (*p<0.001; p<0.01). Evoked currents were measured by two-electrode voltage clamp technique at a holding potential of −70 mM. Black bars indicate time of agonist application. Error bars represent SEM.
	Figure 1. Detection of the hTAAR5 receptor protein.Expression of the rhodopsin-tagged hTAAR5 receptor in transfected, fixed HANA3A cells was detected by the anti-rhodopsin antibody 4D2 and a secondary antibody labeled with the fluorescent dye Alexa Fluor 488 (green). Cell nuclei were stained by DAPI (blue). Left: Cells transfected with hTAAR5, right: mock-transfected control cells. Scaling bar: 20 µm.