The Diacylglycerol Analogs OAG and DOG Differentially Affect Primary Events of Pheromone Transduction in the Hawkmoth Manduca sexta in a Zeitgebertime-Dependent Manner Apparently Targeting TRP Channels.

For the hawkmoth Manduca sexta accumulating evidence suggests that pheromone transduction acts via a metabotropic signal transduction cascade, with G-protein-dependent phospholipase C (PLC) activations generating diacylglycerol (DAG) and inositol trisphosphate as the primary events in hawkmoth pheromone transduction. In contrast, ionotropic olfactory receptor (OR) coreceptor (Orco)-dependent mechanisms do not appear to be involved. In hawkmoths pheromones activated a specific sequence of PLC-dependent ion channels of unknown identity. In several sensory systems transient receptor potential (TRP) ion channels were found downstream of PLC as primary transduction channels. Also in the mammalian vomeronasal organ, DAG-dependent TRP channels are employed. Therefore, we hypothesized that TRPs may be downstream targets for DAG also in the hawkmoth pheromone signal transduction pathway. To test this, we employed two DAG analogs, OAG and DOG for in vivo single-sensillum tip-recordings of pheromone-sensitive sensilla. Since olfactory receptor neurons (ORNs) expressed circadian changes in sensitivity throughout the day, we recorded at two different Zeitgebertimes (ZTs), the hawkmoths activity phase at ZT 1 and its resting phase at ZT 9. We found that the DAG analogs targeted at least two different TRP-like channels that underlie the primary events of hawkmoth pheromone transduction daytime-dependently. At both ZTs OAG sped up and increased the Orco-independent phasic action potential response without affecting the Orco-dependent late, long-lasting pheromone response. Thus, OAG most likely opened a transient Ca2+ permeable TRP channel that was available at both ZTs and that opened pheromone-dependently before Orco. In contrast, DOG slowed down and decreased the sensillum potential, the phasic-, and the late, long-lasting pheromone response. Therefore, DOG appeared to activate a protein kinase C (PKC) that closed TRP-like Ca2+ permeable channels and opened Ca2+ impermeable cation channels, which have been previously described and are most abundant at ZT 9. These data support our hypothesis that hawkmoth pheromone transduction is mediated by metabotropic PLC-dependent mechanisms that activate TRP-like channels as the primary event of pheromone transduction. In addition, our data indicate that at different times of the day different second messenger-dependent ion channels are available for pheromone transduction cascades.

No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta.

Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.

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

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 Cß. 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.