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

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Data_Sheet_1_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.docx

<p>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.</p

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

Scheme of Orco functions in hawkmoth olfactory sensilla (modified after review: [2]).

<p>MsexOrco is suggested to play no role for the primary events of odor/pheromone transduction. In the dendritic cilia Orco acts as “chaperon”, locating and maintaining odor-binding olfactory receptors (OR_x) in membranes of cilia, possibly Ca²⁺/calmoduline-dependently, as shown recently [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.ref049" target="_blank">49</a>]. Odor/pheromone binding to ORs activates a phospholipase (PLCβ) that hydrolyzes phospholipids (PIP₂). The metabotropic odor/pheromone transduction generates rises in IP₃ which open first a Ca²⁺ channel. The rise in intracellular Ca²⁺ starts a cascade of ion channel openings (not shown) that generate the depolarizing receptor potential. The resulting odor-dependent voltage-changes gate Orco in the soma of the olfactory receptor neurons. There, MsexOrco acts as pacemaker channel that controls membrane potential oscillations and, thus, spike threshold and response kinetics. It remains to be studied whether MsexOrco is also gated via cAMP and cGMP, or via protein kinase C-dependent phosphorylation. BL basal lamina, CI cilium, CU cuticle, D dendrite, GL glia, HL hemolymph, ORN olfactory receptor neuron, P pore, RE receptor lymph, TE thecogen cell, TO tormogen cell, TR trichogen cell.</p

Statistics for bombykal responses.

<p>Statistics for bombykal responses.</p

The latency of the first bombykal (BAL)-dependent AP was prolonged by all compounds tested.

<p>(A,B) The amiloride HMA was significantly more potent than OLC15 and MIA at both Zeitgebertimes. Box plots with whiskers from 5 to 95 percentiles. Different scales were used with interrupted error bars to improve illustration of differences. Significant differences are indicated by asterisks (exact P-values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.t002" target="_blank">Table 2</a>; <i>*P</i><0.05; <i>**P</i><0.01; <i>***P</i><0.001).</p

Statistics for current injection experiments.

<p>Statistics for current injection experiments.</p

Orco is a voltage-dependent ion channel controlling spontaneous activity mainly during the hawkmoth´s activity phase.

<p>In tip-recordings of non-stimulated pheromone-sensitive trichoid sensilla the spontaneous action potential (AP) activity within 60 s after current injection (10 s, 3 nA, yellow) was increased more strongly during activity- (A) than during rest-phases (B) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.t003" target="_blank">Table 3</a>). During the activity phase voltage-dependent rise in spontaneous activity was primarily mediated via Orco, since OLC15 prevented it (A). However, Orco seems to play a minor role in mediating the voltage-dependent rise in spontaneous activity during rest (B). Box plots with whiskers from 5 to 95 percentiles. Significant differences are indicated by asterisks (exact P-values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.t003" target="_blank">Table 3</a>; n.s. = not significant; <i>*P</i><0.05; <i>***P</i><0.001).</p

Orco antagonist OLC15 blocks <i>Manduca sexta</i> Orco <i>in vivo</i>.

<p>(A,B) In tip-recordings of pheromone-sensitive trichoid sensilla in intact hawkmoths, Orco agonist VUAA1 (10 μM) increased spontaneous activity of unstimulated, pheromone-sensitive olfactory receptor neurons, as compared to controls (DMSO). During the activity phase (A) application of VUAA1 was significantly more effective than at rest (B) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.t001" target="_blank">Table 1</a>). Co-application of OLC15 dose-dependently (1, 10, 100 μM) interferes with VUAA1-dependent activation of Orco. About half of the ORNs maximal activity was blocked with an intermediate concentration of 10 μM OLC15. Box plots with whiskers from 5 to 95 percentiles. Significant differences are indicated by asterisks (for exact P-values see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166060#pone.0166060.t001" target="_blank">Table 1</a>; n.s. = not significant; <i>**P</i><0.01; <i>***P</i><0.001).</p