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In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

Nolte A, Funk NW, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M - PLoS ONE (2013)

Bottom Line: Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms.We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta.Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology, University of Kassel, Kassel, Germany.

ABSTRACT
The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cβ. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

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Heterologously expressed MsexOrco is activated by VUAA1 and increases spontaneous activity.Furthermore, MsexOrco appears to interact with MsexORs and/or SNMP-1 in heterologous expression systems. (A) Normalized calcium imaging data of HEK 293 cells transiently transfected with MsexOrco. Data for 100 cells are shown, where each line represents the percentage deviation of the fluorescence ratio (Δ(F340/F380)) for one cell. After VUAA1 application (100 µl of 100 µM, arrow) eight of 100 cells show an increase in the fluorescence ratio. (B) Percentages of active cells either transiently transfected with MsexOrco (+Orco) or not (-Orco) after VUAA1 application or without application (spontaneous) are compared (n = number of experiments; for each experiment the percentage of active cells was determined). (C) Box plots show the mean increase in the free intracellular Ca2+ concentration (Δ[Ca2+]i) after VUAA1 application. HEK 293 cells were either not transfected or stably transfected with MsexOrco and optionally cotransfected with MsexSNMP-1 and MsexOR-1, or MsexOR-4 (n = number of cells). (B,C) Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
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pone-0062648-g001: Heterologously expressed MsexOrco is activated by VUAA1 and increases spontaneous activity.Furthermore, MsexOrco appears to interact with MsexORs and/or SNMP-1 in heterologous expression systems. (A) Normalized calcium imaging data of HEK 293 cells transiently transfected with MsexOrco. Data for 100 cells are shown, where each line represents the percentage deviation of the fluorescence ratio (Δ(F340/F380)) for one cell. After VUAA1 application (100 µl of 100 µM, arrow) eight of 100 cells show an increase in the fluorescence ratio. (B) Percentages of active cells either transiently transfected with MsexOrco (+Orco) or not (-Orco) after VUAA1 application or without application (spontaneous) are compared (n = number of experiments; for each experiment the percentage of active cells was determined). (C) Box plots show the mean increase in the free intracellular Ca2+ concentration (Δ[Ca2+]i) after VUAA1 application. HEK 293 cells were either not transfected or stably transfected with MsexOrco and optionally cotransfected with MsexSNMP-1 and MsexOR-1, or MsexOR-4 (n = number of cells). (B,C) Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).

Mentions: In Ca2+ imaging experiments stimulation with 100 µM VUAA1 increased intracellular Ca2+ concentrations in HEK 293 cells transiently transfected with MsexOrco (Fig. 1A). We confirmed that VUAA1 is an MsexOrco agonist since significantly more MsexOrco transfected cells (median: 3%) showed VUAA1-dependent intracellular Ca2+ concentration increases compared to controls not transfected with MsexOrco (median: 1%; Fig. 1B). Additionally the percentage of MsexOrco transfected cells showing VUAA1-dependent Ca2+ concentration increases was significantly higher than the percentage showing spontaneous Ca2+ concentration increases (P = 0.024; median: 1.78%). Moreover, MsexOrco transfected cells showed significantly more spontaneous Ca2+ concentration increases (P = 0.003) than non-Orco transfected cells (median: 0%). From these measurements we conclude that MsexOrco forms a leaky Ca2+-permeable ion channel, whose open-probability can be increased by VUAA1.


In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

Nolte A, Funk NW, Mukunda L, Gawalek P, Werckenthin A, Hansson BS, Wicher D, Stengl M - PLoS ONE (2013)

Heterologously expressed MsexOrco is activated by VUAA1 and increases spontaneous activity.Furthermore, MsexOrco appears to interact with MsexORs and/or SNMP-1 in heterologous expression systems. (A) Normalized calcium imaging data of HEK 293 cells transiently transfected with MsexOrco. Data for 100 cells are shown, where each line represents the percentage deviation of the fluorescence ratio (Δ(F340/F380)) for one cell. After VUAA1 application (100 µl of 100 µM, arrow) eight of 100 cells show an increase in the fluorescence ratio. (B) Percentages of active cells either transiently transfected with MsexOrco (+Orco) or not (-Orco) after VUAA1 application or without application (spontaneous) are compared (n = number of experiments; for each experiment the percentage of active cells was determined). (C) Box plots show the mean increase in the free intracellular Ca2+ concentration (Δ[Ca2+]i) after VUAA1 application. HEK 293 cells were either not transfected or stably transfected with MsexOrco and optionally cotransfected with MsexSNMP-1 and MsexOR-1, or MsexOR-4 (n = number of cells). (B,C) Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
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pone-0062648-g001: Heterologously expressed MsexOrco is activated by VUAA1 and increases spontaneous activity.Furthermore, MsexOrco appears to interact with MsexORs and/or SNMP-1 in heterologous expression systems. (A) Normalized calcium imaging data of HEK 293 cells transiently transfected with MsexOrco. Data for 100 cells are shown, where each line represents the percentage deviation of the fluorescence ratio (Δ(F340/F380)) for one cell. After VUAA1 application (100 µl of 100 µM, arrow) eight of 100 cells show an increase in the fluorescence ratio. (B) Percentages of active cells either transiently transfected with MsexOrco (+Orco) or not (-Orco) after VUAA1 application or without application (spontaneous) are compared (n = number of experiments; for each experiment the percentage of active cells was determined). (C) Box plots show the mean increase in the free intracellular Ca2+ concentration (Δ[Ca2+]i) after VUAA1 application. HEK 293 cells were either not transfected or stably transfected with MsexOrco and optionally cotransfected with MsexSNMP-1 and MsexOR-1, or MsexOR-4 (n = number of cells). (B,C) Significant differences are indicated by asterisks (n.s. = not significant; *P<0.05, **P<0.01, ***P<0.001; Mann-Whitney test).
Mentions: In Ca2+ imaging experiments stimulation with 100 µM VUAA1 increased intracellular Ca2+ concentrations in HEK 293 cells transiently transfected with MsexOrco (Fig. 1A). We confirmed that VUAA1 is an MsexOrco agonist since significantly more MsexOrco transfected cells (median: 3%) showed VUAA1-dependent intracellular Ca2+ concentration increases compared to controls not transfected with MsexOrco (median: 1%; Fig. 1B). Additionally the percentage of MsexOrco transfected cells showing VUAA1-dependent Ca2+ concentration increases was significantly higher than the percentage showing spontaneous Ca2+ concentration increases (P = 0.024; median: 1.78%). Moreover, MsexOrco transfected cells showed significantly more spontaneous Ca2+ concentration increases (P = 0.003) than non-Orco transfected cells (median: 0%). From these measurements we conclude that MsexOrco forms a leaky Ca2+-permeable ion channel, whose open-probability can be increased by VUAA1.

Bottom Line: Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms.We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta.Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Physiology, University of Kassel, Kassel, Germany.

ABSTRACT
The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cβ. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

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