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. ppk25, a Drosophila 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. 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 (Lin, 2005).

As in most other animals, pheromones play key roles in the regulation of sexual behaviors of Drosophila. In particular, several pheromones modulate male courtship of the female, which involves a stereotyped series of behaviors. By analogy with olfactory and gustatory perception of organic molecules in both insects and vertebrates, perception of these pheromones most likely involves interactions with seven-transmembrane receptors and subsequent activation of a G protein-coupled signal transduction pathway. Indeed, a male-specific member of the seven-transmembrane gustatory receptor family, GR68a, has been identified as a putative receptor for female courtship-stimulating pheromones (Bray, 2003). In contrast, gustatory perception of hydrogen and sodium ions, perceived as sour and salty tastes, respectively, has been suggested to involve direct gating of sodium channels of the DEG/ENaC family (Lin, 1999; Lin, 2002). In support of this possibility, inactivation of either ppk11 or ppk19, two Drosophila DEG/ENaC subunit genes, results in loss of behavioral and electrophysiological responses to salt (Liu, 2003; Lin, 2005).

CheB42a, a member of a recently discovered family of Drosophila proteins, is only expressed in a small subset of gustatory sensilla on the front legs of males, suggesting that it may be involved in male-specific gustatory perception (Xu, 2002). Subsequently, Gr68a, a gustatory receptor gene, was found to be expressed in a similar pattern (Bray, 2003). The loss of male response to female pheromones upon either inactivation of Gr68a-expressing neurons or knock-down of Gr68a expression suggests that Gr68a may be a receptor for female pheromones that activate male courtship behavior. CheB42a and Gr68a are expressed in the same subset of gustatory sensilla on male front legs, suggesting that CheB42a also plays a role in this process. Intriguingly, ppk25, a gene predicted to encode another protein with a function in chemical senses, is found only 103 nt downstream of the 3' end of CheB42a (Lin, 2005).

Two deletions were generated that inactivate both CheB42a and ppk25: Delta5-2 and Delta5-22. Males homozygous for either deletion display a much reduced response to females but no similar decrease in other behaviors. In contrast, another deletion that results in complete loss of CheB42a expression but has no effect on ppk25 does not reduce male courtship behavior. A genomic fragment that includes both CheB42a and ppk25 rescues the response of Delta5-22 homozygous males to females, whereas an almost identical fragment lacking ppk25 does not. ppk25^PB, an independent mutation resulting from insertion of a transposable element into the second intron of ppk25, affects male response to females even more severely than Delta5-22, even though this allele has no detectable effect on CheB42a expression. Indeed, ppk25^PB has dominant-negative effects on male response to females, observable both in the presence or absence of a wild-type copy of ppk25. The dominant-negative properties of ppk25^PB are readily interpreted in light of the predicted generation in this mutant of a truncated Ppk25 protein retaining the N-terminal cytoplasmic domain, the first transmembrane domain, and part of the extracellular domain of the normal Ppk25. Similarly truncated variants of various members of the DEG/ENaC family, including several Drosophila ppks, also have dominant-negative properties (Lin, 2005).

The discovery of a role for ppk25 in male response to female pheromones was the unexpected result of an interest in the neighboring CheB42a. The data in this report show that deletion of CheB42a does not decrease overall male response to courtship-activating pheromones. However, the restricted expression of CheB42a in the same subset of gustatory sensilla that express Gr68a and are required for response to female courtship-activating pheromones (Bray, 2003) suggests that CheB42a's requirement may be obscured by functional redundancy with one or more of the other 11 Drosophila CheB genes or, alternatively, that CheB42a has a different role in male-specific chemical senses (Lin, 2005).

Is it a coincidence that two genes implicated in male-specific chemical senses are within ~103 nt of each other? These two genes produce mRNAs of different sizes with related, albeit different, expression patterns. Both are preferentially expressed in male gustatory appendages starting late in pupal development and remaining through at least sexual maturity of the adult males. However, whereas CheB42a is only expressed in male front legs (Xu, 2002), ppk25 mRNA is present at similar levels in legs and in the third antennal segment, and at much lower but detectable levels in heads and bodies. The proximity of these two genes may therefore reflect a shared dependence on regulatory elements important for overlapping spatial and/or temporal characteristics of their expression. Indeed, the lack of detectable ppk25 mRNA in males homozygous for Delta5-2 suggests the presence of a regulatory element essential for ppk25 expression within or immediately downstream of the 3' half of CheB42a. Alternatively, the proximity between these two genes may be more a reflection of their involvement in evolutionarily important and related aspects of sexual behavior (Lin, 2005).

Why can't ppk25 mutant males respond to females normally? Vision and pheromone detection have both been implicated in the response of Drosophila males to females. Absence of visible light or mutations that cause partial or complete blindness reduce, but do not eliminate, male response to females. In addition, a number of studies suggest that males detect courtship-stimulating female pheromones by using either gustation, olfaction, or both chemical senses. Although both vision and olfactory detection of pheromones are important for initiation of courtship behavior, gustatory perception of the same or other pheromones may be required for efficient progression to later steps in the courtship sequence. Because both initiation and maintenance of courtship bouts are affected in dominant-negative as well as null mutations in ppk25, this gene may be required for detection of pheromones by both sensory modalities, a possibility supported by the expression of ppk25 in both olfactory (antennae) and gustatory (wings and legs) appendages (Lin, 2005).

Is a Ppk25-containing sodium channel involved in the peripheral detection of female pheromones? Here the data strongly support the requirement for ppk25 in the male's ability to respond to female courtship-activating pheromones. In addition, mutations in ppk25 do not similarly impair other behaviors that are either largely independent of sensory inputs, such as walking and preening, or sensory-driven such as geotaxis and chemosensory response to sugars. Most importantly, these mutations have no effect on the initiation of courtship behavior in the presence of visible light. Therefore, ppk25's requirement for male response to pheromones likely reflects a specific role in the sensory detection of pheromones or subsequent processing within the central nervous system rather than a more general requirement for neural function or even for performance of courtship behavior. Finally, ppk25 expression is first detectable during late pupal stages, after determination of all of the various types of chemosensory cells and as they undergo the final stages of differentiation, suggesting that ppk25 is required for the function, rather than the development of chemosensory organs (Lin, 2005).

Is ppk25 required in peripheral olfactory or gustatory neurons that sense and respond to female pheromones in the environment or in central nervous system neurons that receive and process the information coming from the periphery? Although these alternatives remain to be tested, the former hypothesis is supported by ppk25's preferential expression in male chemosensory appendages as well as by the established roles of other DEG/ENaC subunits in peripheral sensory responses to mechanical stimuli and salt. ppk25's putative role in pheromone detection may not involve direct participation in the primary molecular response to pheromones. However, recent imaging of the electrophysiological response in mechanosensory neurons indicate that the C. elegans DEG/ENaC gene mec-4 is specifically required for the mechanosensory function (O'Hagan, 2005) rather than the general physiology of the neurons in which it is expressed. Similar questions arise regarding the role ppk25 plays in male detection of female pheromones and in particular, whether it interacts, directly or indirectly, with the G protein-coupled signal transduction pathways that underlie chemical senses in Drosophila as in other animals (Lin, 2005).

Finally, the dominant-negative properties of the ppk25^PB allele most likely reflect the participation of the Ppk25 protein in a multisubunit protein complex. Proteins of the DEG/ENaC family are thought to interact in the formation of heteromeric sodium channels. Several truncated versions of DEG/ENaC proteins have dominant-negative properties that most likely result from their ability to form partial and inactive complexes with other DEG/ENaC subunits. By analogy, the results suggest that one or more of the ~30 other Ppk proteins encoded in the Drosophila genome interacts with Ppk25 within a heteromeric sodium channel. In conclusion, the data demonstrate a role for a member of the DEG/ENaC family of sodium channel subunits in the peripheral detection or central processing of a pheromonal signal. This finding opens the door to the dissection of ppk25's role in pheromone response and its relationship with other proteins involved in pheromone response. Finally, this work suggests that members of the Drosophila ppk family, as well as DEG/ENaC subunits in other organisms, play more complex roles in chemical senses than previously suspected (Lin, 2005).

GENE STRUCTURE

Exons - 6

PROTEIN STRUCTURE

Amino Acids - 451

Structural Domains

The ~30 members of the Drosophila ppk family of genes are part of the large family of DEG/ENaC sodium channel subunits that is found in all animals, from Caenorhabditis elegans to humans, and is involved in a wide variety of functions. Several Drosophila ppks are expressed in gustatory neurons, and ppk11 and ppk19 are required for gustatory response to salt. Family members share common topology, such that they span the membrane twice and have intracellular N- and C-termini; a large extracellular loop includes a conserved cysteine-rich region. DEG/ENaC channels have been implicated a broad spectrum of cellular functions, including mechanosensation, proprioception, pain sensation, gametogenesis, and epithelial Na+ transport. These channels exhibit diverse gating properties, ranging from near constitutive opening to rapid inactivation (Lin, 2005; Mano, 1999).

date revised: 12 November 2005

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