A Drosophila DEG/ENaC channel subunit is required for male response to female pheromones

Abstract

Odorants and pheromones as well as sweet- and bitter-tasting small molecules are perceived through activation of G protein-coupled chemosensory receptors. In contrast, gustatory detection of salty and sour tastes may involve direct gating of sodium channels of the DEG/ENaC family by sodium and hydrogen ions, respectively. We have found that ppk25, a Drosophila melanogaster gene encoding a DEG/ENaC channel subunit, is expressed at highest levels in the male appendages responsible for gustatory and olfactory detection of female pheromones: the legs, wings, and antennae. Mutations in the ppk25 gene reduce or even abolish male courtship response to females in the dark, conditions under which detection of female pheromones is an essential courtship-activating sensory input. In contrast, the same mutations have no effect on other behaviors tested. Importantly, ppk25 mutant males that show no response to females in the dark execute all of the normal steps of courtship behavior in the presence of visible light, suggesting that ppk25 is required for activation of courtship behavior by chemosensory perception of female pheromones. Finally, a ppk25 mutant allele predicted to encode a truncated protein has dominant-negative properties, suggesting that the normal Ppk25 protein acts as part of a multiprotein complex. Together, these results indicate that ppk25 is necessary for response to female pheromones by D. melanogaster males, and suggest that members of the DEG/ENaC family of genes play a wider role in chemical senses than previously suspected.

Fig. 1.

The CheB42a and ppk25 genes: transposon insertions and deletions. (a) Map showing the genomic region that includes the CheB42a and ppk25 genes [modified from Flybase (46), http://flybase.org]. The insertion sites for two transposable elements used in this study, a P-element in line KG05881 (9) and a Piggyback element in line e04217 (28), are indicated by arrows. The CheB42a transcription unit is based on the sequence of the corresponding cDNA (10). The predicted transcription unit of ppk25 is supported by the sequence similarity of the predicted protein to other Drosophila Ppks (8), and the position of the introns confirmed by direct sequence of RT-PCR products (data not shown). (b) The endpoints of the Δ5-68, Δ5-2, and Δ5-22 deletions as well as the presence of a partial P element remaining in Δ5-22 are indicated. (c) Cartoon comparing the structural domains present in a wild-type Ppk25 protein and the truncated Ppk25^PB predicted to result from insertion of a Piggyback transposable element in the second intron of the ppk25 gene (see text). TM1 and TM2, transmembrane domains 1 and 2; ICD, intracellular domain; ECD, extracellular domain.

Fig. 2.

ppk25 mRNA is preferentially expressed in appendages highly enriched in chemosensory organs. mRNA extracted from appendages (third antennal segment, legs and wings), heads (without third antennal segment), and bodies (without heads, legs or antennae) was analyzed on a Northern blot that was hybridized with a full-length ppk25 cDNA probe and exposed for 48 h (Left) or six days (Center Left). The same filter was subsequently boiled and rehybridized with a CheB42a probe. A third hybridization with a probe for the ubiquitous rp49 mRNA (47) shows that somewhat less “Appendages” mRNA was loaded compared to the other body parts so that preferential ppk25 expression in appendages is underrepresented. A, appendages; B, bodies; H, heads.

Fig. 3.

ppk25 is expressed in adult appendages involved in taste and smell. Real-time PCR was performed on cDNA prepared from RNA as follows. (a) RNA extracted from male or female body parts as in Fig. 2. In three independent experiments, expression of ppk25 was higher in males than female appendages with an average ratio of 2.4 ± 0.46 (standard error). (b) RNA extracted from single types of male appendages. In three independent experiments, expression in antennae was within a factor of two of that found in combined legs and wings. App., appendages. (c) RNA extracted from whole animals at specific developmental stages. In two independent experiments, ppk25 expression was observed in dark pupae and young adults but not larvae or light pupae. L1 + 2, first and second instar larvae; L3, third instar larvae; l.p., light pupae; d.p., dark pupae; 1 d.o. and 3 d.o., 1- and 3-day-old adults, respectively. In a and b, the relative concentration of ppk25 mRNA is obtained by dividing the normalized value for each sample (see Experimental Procedures) by the lowest value observed in the same experiment (for example, in a, female bodies are set at 1). In c, because the expression level of ppk25 in larvae and light pupae is below detection, the highest sample (dark pupae) was set at 1.

Fig. 4.

Three deletions that remove part or all of the CheB42a gene have differential effects on ppk25 expression. (a) Western blot of extracts from the front legs of males of each genotype using an anti-CheB42a antibody (10). The CheB42a protein is absent in all three deletions. (b) Δ5-68, Δ5-2, and Δ5-22 have differential effects on ppk25 mRNA. Poly(A)+ mRNA was extracted from the appendages of male flies homozygous for each of the deletions and a control, G7, and analyzed on a Northern blot that was sequentially probed with radiolabelled full-length ppk25 (Upper) and rp49 (Lower) cDNAs. RT-PCR experiments confirm that the mRNAs expressed in Δ5-22 males initiate within the P-element sequences that remain in that deletion and proceed through the remaining ppk25 sequences (not shown). Given that Δ5-22 retains normal sequences up to 70 bp upstream of the 5′ splice site for the third intron of ppk25, we were surprised to find that this deletion specifically disrupts splicing of intron 3 but not that of intron 4 (Experimental Procedures). Importantly, although these hybrid mRNAs contain sequences encoding ppk25 C-terminal residues, retention of intron 3 disrupts all but 23 aa of the remaining ppk25 ORF within a poorly conserved stretch of the Ppk25 extracellular domain. Furthermore, the ATG that initiates this residual ppk25 ORF is unlikely to function as an initiation of translation because it follows, by 25 nt, another ATG that has a better match to the Kozak consensus translation initiation site (48) (data not shown). Together, these results suggest that no Ppk25-related peptide is produced in Δ5-22 homozygous flies.

Fig. 5.

Male response to females is debilitated by deletions that remove or prevent expression of ppk25, and is restored by a ppk25-carrying transgene. The response of males of different genotypes to females was quantitated by a courtship index: the fraction of the observation time spent performing any step in the courtship sequence multiplied by 100 (26), and similar indices measure the time spent walking and preening. (a) Courtship response is dramatically reduced in males homozygous for Δ5-2 or Δ5-22, but not Δ5-68, relative to G7 isogenic control males (P < 9 × 10^-4 and P < 2 × 10^-4 for Δ5-2 and Δ5-22, respectively). (b) Introduction of Tg1, a transgene carrying the genomic region that includes CheB42a and ppk25, rescues the courtship response of Δ5-22 homozygous males (+Tg1), whereas Tg2, an almost identical transgene that lacks ppk25 (+Tg2), does not. Error bars indicate standard error of the mean and n for each genotype is indicated immediately above.

Fig. 6.

The ppk25^PB allele has a dominant-negative effect on male response to female pheromones. The male response to females was measured as in Fig. 5. The males tested carry the following mutations: ppk25^PB, Piggyback insertion into ppk25 (line e04217); C^PB, control with a normal ppk25 gene and the same Piggyback element inserted at an unrelated site on the second chromosome (line e00673); Δ42E, a deletion spanning 20 genes in the ppk25 region [Df(2R)Exel6051]; C^Δ, a control deletion in an unrelated region on the second chromosome that retains a normal ppk25 gene (Df(2R)ED1552). ^*1 and ^*2, P = 3 × 10^-5 and P = 0.012 for comparisons of the control C^PB/Δ42E to ppk25^PB/Δ42E and ppk25^PB/C^Δ, respectively.

Fig. 7.

Visible light enables courtship behavior in males carrying the dominant-negative ppk25^PB allele. Male response to females was measured as in Fig. 5 except for the presence of visible light. For this experiment, three separate parameters of male behavior are shown to demonstrate the differential effect of the dominant-negative ppk25^PB allele: lag to courtship, number of courtship bouts per minute, and length of courtship bouts (see text). The males tested carry a Δ5-22 deletion on one copy of the second chromosome and are completely isogenic except for the presence on their other second chromosome of either (i) the dominant-negative ppk25^PB allele, or (ii) the normal ppk25 gene and another Piggyback insertion at an unrelated site. In the presence of visible light, replacement of the normal ppk25 gene by the ppk25^PB allele causes a statistically significant decrease in the average length of a courtship bout (P < 0.02) but no change in the lag to courtship or in the number of courtship bouts per minute.