Identified, with all of them having been annotated previously in dbSNP

Identified, with all of them having been annotated previously in dbSNP (Table 1). Additional allele frequency data based on the genotypes of 4,300 European American individuals was pulled from the NHLBI Grand Opportunity Exome Sequencing Project’s (ESP) Exome Variant Server, release ESP6500 [21]. In general, allele frequency estimates based on the Emixustat (hydrochloride) control pool data aligned well with frequencies from the ESP dataset (Table 1). Analyzing hTAAR genes from TMA anosmics. A second round of sequencing included seven subjects with a specific anosmia for trimethylamine (TMA). The coding regions of the six putatively functional human TAAR genes (hTAAR1, 22, 25, 26, 28 and 29) were sequenced individually by Sanger sequencing. In total, seven SNPs were found in at least one of the anosmic subjects (Table 1). All of the identified SNPs were also present in the control pool. In the hTAAR5 gene, two SNPs were found. However, both of them are synonymous and do not occur at a significantly different frequency in the anosmics’ group compared to the control pool (374913-63-0 web Fisher’s Exact Test, p.0.05). A lossof-function variant in the hTAAR9 gene (rs2842899) was moreHuman TAAR5 Is Activated by TrimethylamineFigure 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 mM. Responses were normalized to the response to forskolin (10 mM). Data are given as mean 6 SEM of 2?0 independent experiments, each performed in duplicates. TMA and DMEA induced signals significantly differing from mock-transfected controls (***p,0.001; **p,0.01). doi:10.1371/journal.pone.0054950.gfrequent in the group of anosmics than in the control pool, but not significantly (p = 0.351, Fisher’s Exact Test). Five SNPs that had been identified in the control pool were not found in the group of anosmics, but the differences in their allele frequencies (assumed to be 0 for the anosmics) were not statistically significant. The same is true for four SNPs found in the hTAAR1 and hTAAR6 genes. These data show that there is no association between a polymorphism in an hTAAR coding sequence and the observed anosmic individuals.DiscussionIn our study, we present the first deorphanization of a human olfactory TAAR. Human TAAR5 can be activated with a high specificity by the tertiary amines TMA (full agonist) 15857111 and DMEA (partial agonist), but not by any other of the tested aminic compounds. Interestingly, TAAR5 ligand specificity differs between humans and mice. The human receptor is exclusively activated by tertiary amines with methyl or ethyl side chains. In contrast, the murine TAAR5 ortholog is also activated by Nmethylpiperidine and the secondary amine dimethylamine [2,11]. The finding that TMA activates the hTAAR5 receptor is of substantial interest because this receptor is the most highly expressed TAAR gene in the 1317923 human OE [12,22]. It is stillFigure 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 mM). EC50 = 116 mM (TMA) and EC50 = 169 mM (DMEA). Detection threshold for TMA is 1 mM (*p,0.05) and for DMEA 30 mM (*p,0.05). Error bars represent SEM. doi:10.1371/journal.pone.0054950.gHuman TAAR5 Is Activated by TrimethylamineFigure 5. TMA and DMEA activate hTAAR5 expressed in Xenopus oocytes. A: IBMX induced currents in oocytes expressing the reporter channel CFTR only. B:.Identified, with all of them having been annotated previously in dbSNP (Table 1). Additional allele frequency data based on the genotypes of 4,300 European American individuals was pulled from the NHLBI Grand Opportunity Exome Sequencing Project’s (ESP) Exome Variant Server, release ESP6500 [21]. In general, allele frequency estimates based on the control pool data aligned well with frequencies from the ESP dataset (Table 1). Analyzing hTAAR genes from TMA anosmics. A second round of sequencing included seven subjects with a specific anosmia for trimethylamine (TMA). The coding regions of the six putatively functional human TAAR genes (hTAAR1, 22, 25, 26, 28 and 29) were sequenced individually by Sanger sequencing. In total, seven SNPs were found in at least one of the anosmic subjects (Table 1). All of the identified SNPs were also present in the control pool. In the hTAAR5 gene, two SNPs were found. However, both of them are synonymous and do not occur at a significantly different frequency in the anosmics’ group compared to the control pool (Fisher’s Exact Test, p.0.05). A lossof-function variant in the hTAAR9 gene (rs2842899) was moreHuman TAAR5 Is Activated by TrimethylamineFigure 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 mM. Responses were normalized to the response to forskolin (10 mM). Data are given as mean 6 SEM of 2?0 independent experiments, each performed in duplicates. TMA and DMEA induced signals significantly differing from mock-transfected controls (***p,0.001; **p,0.01). doi:10.1371/journal.pone.0054950.gfrequent in the group of anosmics than in the control pool, but not significantly (p = 0.351, Fisher’s Exact Test). Five SNPs that had been identified in the control pool were not found in the group of anosmics, but the differences in their allele frequencies (assumed to be 0 for the anosmics) were not statistically significant. The same is true for four SNPs found in the hTAAR1 and hTAAR6 genes. These data show that there is no association between a polymorphism in an hTAAR coding sequence and the observed anosmic individuals.DiscussionIn our study, we present the first deorphanization of a human olfactory TAAR. Human TAAR5 can be activated with a high specificity by the tertiary amines TMA (full agonist) 15857111 and DMEA (partial agonist), but not by any other of the tested aminic compounds. Interestingly, TAAR5 ligand specificity differs between humans and mice. The human receptor is exclusively activated by tertiary amines with methyl or ethyl side chains. In contrast, the murine TAAR5 ortholog is also activated by Nmethylpiperidine and the secondary amine dimethylamine [2,11]. The finding that TMA activates the hTAAR5 receptor is of substantial interest because this receptor is the most highly expressed TAAR gene in the 1317923 human OE [12,22]. It is stillFigure 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 mM). EC50 = 116 mM (TMA) and EC50 = 169 mM (DMEA). Detection threshold for TMA is 1 mM (*p,0.05) and for DMEA 30 mM (*p,0.05). Error bars represent SEM. doi:10.1371/journal.pone.0054950.gHuman TAAR5 Is Activated by TrimethylamineFigure 5. TMA and DMEA activate hTAAR5 expressed in Xenopus oocytes. A: IBMX induced currents in oocytes expressing the reporter channel CFTR only. B:.