Ogously expressed hTAAR5 using Xenopus laevis oocytes, and screened hTAAR5 with various amines, focusing on DMEA and TMA. This system was used for h/mTAAR1 [1,15] and mammalian odorant receptors and employs CFTR as a AZP-531 web reporter channel [16,17], necessary for the induction of currents (Materials and methods). As a control for CFTR expression level, each oocyte was tested for its sensitivity to the phosphodiesterase inhibitorFigure 1. Detection of the hTAAR5 receptor protein. Expression of the rhodopsin-tagged hTAAR5 receptor in transfected, fixed buy AZP-531 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 mm. doi:10.1371/journal.pone.0054950.gHuman TAAR5 Is Activated by TrimethylamineFigure 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) Nmethylpiperidine, (10) pyridine, (11) b-phenylethylamine, (12) skatole, (13) ethanolamine, (14) putrescine, (15) isobutylamine, (16) dimethylbutylamine. doi:10.1371/journal.pone.0054950.gisobutylmethylxantine (IBMX, 1 mM), which induces a rise in intracellular cAMP and subsequently CFTR mediated inward currents. Human TAAR5 was tested for a total of 10 different amines: b-phenylethylamine, tyramine, serotonin, isobutylamine, TMA, DMEA, N-methylpiperidine, putrescine, cyclohexylamine and ethanolamine, all applied at a concentration of 100 mM. TMA and DMEA induced inward currents on oocytes injected with hTAAR5 but failed to induce any currents in oocytes expressing the reporter channel only (Fig. 5A,B). Mean currents were higher for TMA (7346221 nA, n = 11) than for DMEA (136656 nA, n = 6), both significantly smaller than the mean currents induced by IBMX (1625619 nA, p,0.05, n = 15). The threshold of TMA detection was 1 mM (Fig. 5C), similar to the Cre-luciferase assay (Fig. 4). Normalized to the IBMX induced currents 100 mM, TMA and DMEA evoked 42.5612.8 and 14.666.0 of the IBMX induced currents respectively (Fig. 5C). None of the other tested amines evoked notable currents. Our Xenopus data confirm that TMA and DMEA are activating ligands for human TAAR5.Characterization of SNPs in hTAAR GenesAmoore identified TMA as the primary fishy odor and found that about 7 of the human population are specifically anosmic for this odorant [18]. For screening a large group of subjects (n = 393) with the primary odorant to find TMA anosmics, we used a standardized test concentration in water that is 16 times threshold [19]. In two different screenings with forced choice tests we identified 12 24786787 TMA anosmics. To figure out if the anosmia is caused by a SNP in an hTAAR coding sequence, especially inhTAAR5, the sequencing data from seven subjects were used for subsequent SNP analysis (see Materials and methods). Reference data. On the Illumina GAIIx platform, the exons of the six putatively functional human TAAR genes (hTAAR1, 22, 25, 26, 28 and 29) were sequenced in a pool of two hundred randomly selected subjects and scanned for putative Single Nucleotide Polymorphisms (SNPs). Allele frequencies of these putative SNPs were calculated using the CRISP algorithm [20]. In total, 12 SNPs were.Ogously expressed hTAAR5 using Xenopus laevis oocytes, and screened hTAAR5 with various amines, focusing on DMEA and TMA. This system was used for h/mTAAR1 [1,15] and mammalian odorant receptors and employs CFTR as a reporter channel [16,17], necessary for the induction of currents (Materials and methods). As a control for CFTR expression level, each oocyte was tested for its sensitivity to the phosphodiesterase inhibitorFigure 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 mm. doi:10.1371/journal.pone.0054950.gHuman TAAR5 Is Activated by TrimethylamineFigure 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) Nmethylpiperidine, (10) pyridine, (11) b-phenylethylamine, (12) skatole, (13) ethanolamine, (14) putrescine, (15) isobutylamine, (16) dimethylbutylamine. doi:10.1371/journal.pone.0054950.gisobutylmethylxantine (IBMX, 1 mM), which induces a rise in intracellular cAMP and subsequently CFTR mediated inward currents. Human TAAR5 was tested for a total of 10 different amines: b-phenylethylamine, tyramine, serotonin, isobutylamine, TMA, DMEA, N-methylpiperidine, putrescine, cyclohexylamine and ethanolamine, all applied at a concentration of 100 mM. TMA and DMEA induced inward currents on oocytes injected with hTAAR5 but failed to induce any currents in oocytes expressing the reporter channel only (Fig. 5A,B). Mean currents were higher for TMA (7346221 nA, n = 11) than for DMEA (136656 nA, n = 6), both significantly smaller than the mean currents induced by IBMX (1625619 nA, p,0.05, n = 15). The threshold of TMA detection was 1 mM (Fig. 5C), similar to the Cre-luciferase assay (Fig. 4). Normalized to the IBMX induced currents 100 mM, TMA and DMEA evoked 42.5612.8 and 14.666.0 of the IBMX induced currents respectively (Fig. 5C). None of the other tested amines evoked notable currents. Our Xenopus data confirm that TMA and DMEA are activating ligands for human TAAR5.Characterization of SNPs in hTAAR GenesAmoore identified TMA as the primary fishy odor and found that about 7 of the human population are specifically anosmic for this odorant [18]. For screening a large group of subjects (n = 393) with the primary odorant to find TMA anosmics, we used a standardized test concentration in water that is 16 times threshold [19]. In two different screenings with forced choice tests we identified 12 24786787 TMA anosmics. To figure out if the anosmia is caused by a SNP in an hTAAR coding sequence, especially inhTAAR5, the sequencing data from seven subjects were used for subsequent SNP analysis (see Materials and methods). Reference data. On the Illumina GAIIx platform, the exons of the six putatively functional human TAAR genes (hTAAR1, 22, 25, 26, 28 and 29) were sequenced in a pool of two hundred randomly selected subjects and scanned for putative Single Nucleotide Polymorphisms (SNPs). Allele frequencies of these putative SNPs were calculated using the CRISP algorithm [20]. In total, 12 SNPs were.
