Amplification of Drosophila Olfactory Responses by a DEG/ENaC Channel

Abstract

Insect olfactory receptors operate as ligand-gated ion channels that directly transduce odor stimuli into electrical signals. However, in the absence of any known intermediate transduction steps, it remains unclear whether and how these ionotropic inputs are amplified in olfactory receptor neurons (ORNs). Here, we find that amplification occurs in the Drosophila courtship-promoting ORNs through Pickpocket 25 (PPK25), a member of the degenerin/epithelial sodium channel family (DEG/ENaC). Pharmacological and genetic manipulations indicate that, in Or47b and Ir84a ORNs, PPK25 mediates Ca²⁺-dependent signal amplification via an intracellular calmodulin-binding motif. Additionally, hormonal signaling upregulates PPK25 expression to determine the degree of amplification, with striking effects on male courtship. Together, these findings advance our understanding of sensory neurobiology by identifying an amplification mechanism compatible with ionotropic signaling. Moreover, this study offers new insights into DEG/ENaC activation by highlighting a novel means of regulation that is likely conserved across species.

Keywords: DEG/ENaC; Ir84a; Or47b; PPK25; courtship behavior; insect olfactory amplification; ionotropic signaling; juvenile hormone; olfactoy receptor neurons.

Figure 1.. ppk25 is required for the amplification of Or47b ORN responses.

(A) Single-sensillum recording. Representative traces (top), raster plots (middle), and peri-stimulus time histograms (PSTH, bottom) are shown for Or47b ORN responses in wildtype males (palmitoleic acid, 10⁻²). Line width: s.e.m. Gray bar: stimulus duration. (B) Dosage curves of Or47b spike responses from 2d and 7d males. Adjusted peak responses (pre-stimulus baseline activity subtracted from the peak response, see Methods). Parallel experiments, mean ± s.e.m. (n=12, from 6~7 flies). *P<0.05, t-test. (C) Confocal image of the antennal lobe. ppk25-GAL4 labeled Or47b and Ir84a ORNs in 7d males (magenta, VAllm and VL2a glomeruli, respectively). nc82: neuropil marker (blue). Scale bar: 10 μm. (D-E) As in (A-B) except that recordings were performed in ppk25 mutant males (ppk25^Δ5-22). (F) Quantification of Or47b ORN responses to palmitoleic acid (10⁻²). Or47b-GAL4 and UAS-ppk25 heterozygous controls (Δppk25/+), Δppk25, another ppk25 mutant allele (Δppk25^LexA) and genetic rescue ofppk25 in the Or47b ORNs. Mean ± s.e.m. (n=9, from 4~6 flies). (G-H) RNAi knockdown of ppk25 reduced Or47b ORN responses in 7d males. Sample traces (G) and dosage curves (H) are shown. *P<0.05, **P<0.005, ***P<0.0005, t-test. (I) Quantification of Or47b ORN responses to palmitoleic acid (10⁻²). Parallel experiments, mean ± s.e.m. (n=10, from 6~8 flies). ANOVA followed by Tukey’s test. Significant differences (P <0.05) are indicated by different letters, for example between the 7d controls (a) and the RNAi group (b, c). No significant differences were observed between groups indicated by the same letters. See also Figures S1 and S2. Table S1 lists genotypes for all experiments.

Figure 2.. ppk25 in Or47b ORNs impacts courtship

(A) Courtship competition assays were conducted with one 3d wildtype female and three 7d males of different genotypes: Or47b-GAL4/+, UAS-ppk25-RNAi/+, and Or47b-GAL4/UAS-ppk25-RNAi. Cumulative copulation rates over time are shown for each genotype. (B) Copulation rates at the end of the 2-hr assays. Lines connect results from the same experiments (n=5, total 102 matches). (C-D) Single-pair courtship assays were conducted with one 3d wildtype female and one 7d male of different genotypes as indicated in (A-B). Courtship latency (C) and courtship index (D) are shown for each genotype (n=13-14). Horizontal bars: mean. Significant differences (P <0.05) are indicated by different letters, ANOVA followed by Tukey’s test.

Figure 3.. Overexpression of ppk25 increases Or47b ORN responses in males.

(A) Quantitative RT-PCR of ppk25 from male antennal mRNA. MTP: methoprene-treated. Expression level is normalized to that of 2d males (n=12; *P<0.05, t-test). (B-C) Overexpression ofppk25 elevated Or47b ORN responses in 2d males. Sample traces, raster, PSTH (palmitoleic acid, 10⁻²) (B) and dosage curves (C) are shown. Adjusted peak responses. *P<0.05, **P<0.005, t-test. (D) Quantification of Or47b ORN responses to palmitoleic acid (10⁻¹) in 2d males. Parallel experiments, mean ± s.e.m. (n=10, from 6~8 flies). (E) Comparison of Or47b ORN responses to palmitoleic acid (10⁻¹) in 7d wildtype, Δppk25/+ heterozygous, and Δppk25 homozygous mutant males. Mean ± s.e.m. (n= 9~12, from 7~9 flies). Significant differences between any two groups (P <0.05) are indicated by different letters, ANOVA followed by Tukey’s test. See also Figures S3, S4 and S5.

Figure 4.. PPK25 is a Ca²⁺-activated transduction channel in Or47b ORNs.

(A) Topology of PPK25. CBM: calmodulin binding motif. (B) Multiple sequence alignment. Conserved PPK25 CBM residues are shaded in gray. (C) Reducing extracellular Ca²⁺ with EGTA attenuated Or47b responses in wildtype (left panels) but not Δppk25 males (right panels). Top and bottom traces indicate LFP and the corresponding spike responses. Black: control responses; magenta: responses after 5-min application of 5 mM EGTA. Horizontal bar: 0.5-s palmitoleic acid (10⁻²). Parallel experiments, line width indicates s.e.m. (n=9, from 5~7 flies). Lines connect results from the same neurons. (D) Similar to (C) except that 500 μm W-7 was used to block CaM (n=7, from 4~6 flies). (E) CaM binding assay with wildtype or mutant CBM peptides. Numbers are signature hydrophobic residues (top panel). Western blotting with CaM antibody (bottom panel). The input, unbound and pull-down fractions are shown. (F) Rescuing ppk25 mutation with wildtype PPK25 and W-7 was used to block CaM (n=5, from 5 flies, right panel). (G) As in (F) except that PPK25 with 1-8-14 CBM mutations was expressed. *P<0.05, **P<0.005, ***P<0.0005, paired t-test. See also Figures S6 and S7.

Figure 5.. Age-dependent response plasticity in Ir84a ORNs.

(A) Single-sensillum recording. Representative traces (top), raster plots (middle), and PSTH (bottom) are shown for Ir84a ORN responses (phenylacetaldehyde, 10⁻²). Line width: s.e.m. Gray bar: stimulus duration. (B) Dosage curves of Ir84a spike responses (adjusted peak responses). (C-D) As in (A-B) except that recordings were performed in Δppk25 males (n=8, from 4~6 flies). (E) Ir84a responses from 2d males treated for two days with solvent (ethanol, control) or methoprene, a synthetic juvenile hormone analog. (F) Dosage curves of Ir84a spike responses (adjusted peak responses). Parallel experiments, mean ± s.e.m. (n=8, from 4~6 flies). (G-H) As in (E-F) except that 2d ppk25 mutant males were examined. Parallel experiments, mean ± s.e.m. (n=6, from 4~6 flies). (I-J) Overexpression of ppk25 elevated Ir84a ORN responses in 2d males. Parallel experiments, mean ± s.e.m. (n=8, from 4~6 flies). (K-L) As in (A-B) except that recordings were performed in Ir84a^Q401R males. Parallel experiments, mean ± s.e.m. (n=12, from 8~10 flies). *P<0.05, **P<0.005, ***P<0.0005, t-test.

Figure 6.. PPK25 also functions as a Ca²⁺-activated transduction channel in Ir84a ORNs.

(A-B) LFP and the corresponding spike responses from wildtype, Δppk25, and Ir84a^Q401R males (7d). Black: control responses; magenta: responses after 5-min application of 5 mM EGTA (A) or 500 μM W-7 (B). Horizontal bar: 0.25-s phenylacetaldehyde (10⁻²) (n=5~10, from 4~10 flies). **P<0.005, ***P<0.0005, paired t-test.

Figure 7.. Age-dependent response plasticity in tarsal ppk25⁺ GRNs.

(A-B) Tip recording of foreleg gustatory sensilla. Representative traces are shown for Tm4c and Tm4b sensilla in response to solvent (1 mM KCl), 7,11-heptacosadiene (7,11-HD, 20 ng/μl) or 7-tricosene (7-T, 10 ng/μl) from 7d wildtype males. (C) Quantification of Tm4c GRN spike responses to 7,11-HD from wildtype, Δppk25, and ppk25-CBM mutant males (n= 8~18, from 6~13 flies). Average spike responses for the first second following tastant presentation are shown. **P<0.005, t-test. (D) As in (C) except that the spike responses to 7-T were recorded (n= 8, from 6~8 flies).