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pickpocket 28: Biological Overview | References

BIOLOGICAL OVERVIEW

The detection of water and the regulation of water intake are essential for animals to maintain proper osmotic homeostasis. Drosophila and other insects have gustatory sensory neurons that mediate the recognition of external water sources (Evans, 1962; Meunier, 2000; Inoshita, 2006), but little is known about the underlying molecular mechanism for water taste detection. This study identified a member of the Degenerin/Epithelial Sodium Channel family (Kellenberger, 2002), ppk28, as an osmosensitive ion channel that mediates the cellular and behavioral response to water. This study used molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons and loss of ppk28 abolishes water sensitivity. Moreover, ectopic expression of ppk28 confers water sensitivity to bitter-sensing gustatory neurons in the fly and sensitivity to hypo-osmotic solutions when expressed in heterologous cells. These studies link an osmosensitive ion channel to water taste detection and drinking behavior, providing the framework for examining the molecular basis for water detection in other animals (Cameron, 2010).

To uncover novel molecules involved in taste detection, a microarray-based screen was perfected for genes expressed in taste neurons. Proboscis RNA from flies homozygous for a recessive poxn null mutation was compared to RNA from heterozygous controls. poxn mutants have a transformation of labellar gustatory chemosensory bristles into mechanosensory bristles, and therefore lack all taste neurons. Whole genome microarray comparisons revealed that 256 of ~18,500 transcripts were significantly decreased in poxn mutants (>2 fold enrichment in control relative to poxn). These included 18 gustatory receptors (representing a 21-fold enrichment in the gene set) and 8 odorant binding proteins (13-fold enrichment) (Cameron, 2010).

In the mammalian gustatory system, ion channels mediate the detection of sour and salt tastes (Yarmolinsky, 2009), suggesting that ion channel genes may also participate in Drosophila taste detection. Therefore the expression pattern of candidate taste-enriched ion channels was examined. The putative promoter of one gene, pickpocket 28 (ppk28), directed robust reporter expression in taste neurons on the proboscis. ppk28 belongs to the Degenerin/Epithelial sodium channel family (Deg/ENaC) and these channels are involved in the detection of diverse stimuli, including mechanosensory stimuli, acids and sodium ions (Kellenberger, 2002). In the brain, ppk28-Gal4 drives expression of GFP in gustatory sensory axons that project to the primary taste region, the subesophageal ganglion. In situ hybridization experiments confirmed that transgenic expression recapitulates that of the endogenous gene, as 48/52 of ppk28-Gal4 neurons expressed endogenous ppk28 (Cameron, 2010).

Previous studies have identified different taste cell populations in the proboscis, including cells labeled by the gustatory receptor Gr5a that respond to sugars and cells marked by Gr66a that respond to bitter compounds. To determine whether these taste neurons express ppk28-Gal4, co-labeling experiments were performed with reporters for Gr5a and Gr66a. These experiments revealed that ppk28 did not co-label Gr5a cells or Gr66a cells, and is thus unlikely to participate in sweet or bitter taste detection. An enhancer-trap Gal4 line, NP1017-Gal4, labels water-sensing neurons in taste bristles on the proboscis (Inoshita, 2006) and carbonation-sensing neurons in taste pegs (Fischler, 2007). ppk28 is expressed in taste bristles but not in taste pegs. Interestingly, ppk28 showed partial co-expression with NP1017-Gal4, with the majority of ppk28-positive cells containing NP1017-Gal4 (22/30). This correlation suggested the intriguing possibility that ppk28 participates in water taste detection (Cameron, 2010).

To directly investigate the response specificity of ppk28-expressing neurons, the genetically encoded calcium sensor G-CaMP was expressed in ppk28-Gal4 cells, the proboscis was stimulated with taste substances, and activation of ppk28-Gal4 projections were monitored in the living fly by confocal microscopy. ppk28-Gal4 neurons were tested with a panel of taste solutions, including sugars, bitter compounds, salts, acids and water. ppk28-Gal4 neurons showed robust activity upon water stimulation. In addition, ppk28-positive cells responded to other aqueous solutions even in the presence of a wide range of chemically distinct compounds. This response diminished as a function of concentration. Taste compounds such as NaCl, sucrose and citric acid significantly decreased the response. In addition, compounds unlikely to elicit taste cell activity such as ribose, a sugar that does not activate Gr5a cells, N-methyl-D-glucamine (NMDG), an impermeant organic cation and the non-ionic high molecular weight polymer polyethylene glycol (PEG, 3350 average molecular weight), all blunted the response in a concentration-dependent manner. These data demonstrate that ppk28-expressing neurons respond to hypo-osmotic solutions. This response profile is consistent with previous electrophysiological studies that identified a class of labellar taste neurons activated by water and inhibited by salts, sugars and amino acids (Inoshita, 2005; Meunier; 2009; Cameron, 2010).

To determine the function of ppk28 in the water response, a ppk28 null mutant was generated by piggybac transposon mediated gene deletion, removing 1.769kb surrounding the ppk28 gene. The water responses of ppk28 control, mutant and rescue flies were examined by extracellular bristle recordings of l-type labellar taste sensilla. These recordings monitor the responses of the four gustatory neurons in a bristle, including water cells and sugar cells. Control flies showed 12.0±0.9 spikes/sec when stimulated with water. Remarkably, ppk28 mutant cells had a complete loss of the response to water (spikes/sec=0.8±0.1). This response was partially rescued by reintroduction of ppk28 into the mutant background (spikes/sec=6.4±1.0), demonstrating that defects were due to loss of ppk28. Responses to sucrose were not significantly different among the three genotypes, arguing that the loss of ppk28 specifically eliminates the water response. These results were confirmed by G-CaMP imaging experiments that monitor the response of the entire ppk28 population. As expected, ppk28-Gal4 neurons in the mutant did not show fluorescent increases to water and transgenic re-introduction of ppk28 rescued the water response. Taken together, the electrophysiological and imaging data demonstrate that ppk28 is required for the cellular response to water (Cameron, 2010).

The detection of water in the environment and the internal state of the animal may both contribute to drive water consumption. To evaluate the degree to which water taste detection contributes to consumption, the behavioral responses were examined of ppk28 control, mutant and rescue flies to water. Drinking time rather than drinking volume was used to monitor consumption due to difficulty in reliably detecting small volume changes. When presented with a water stimulus, control flies drank on average 10.3±1.1 seconds, mutants drank 3.0±0.3 seconds and rescue flies drank 11.5±1.5 seconds. Additionally, control, mutant and rescue flies ingested sucrose equally, showing that ppk28 mutants do not have general drinking defects. Similar defects in water detection were seen when control, mutant and rescue flies were tested on the proboscis extension reflex to water or when genetically ablating ppk28-Gal4 neurons. Although ppk28 mutants lack water taste cell responses and drink less, they still do consume water, arguing that additional mechanisms must exist to ensure water uptake. These experiments reveal that water taste neurons are necessary for normal water consumption. Moreover, they establish a link between water taste detection in the periphery and the drive to drink water (Cameron, 2010).

Whether ppk28 is directly involved in water detection was examined. If ppk28 is the water sensor, then its expression in non-water sensing cells should bestow responsiveness to water. To test this, the Gal4/UAS system was used to ectopically express ppk28 in Gr66a-expressing, bitter-sensing neurons, and taste-induced responses were monitored by extracellular bristle recordings and G-CaMP imaging experiments. For extracellular bristle recordings, responses were recorded from i-type sensilla which contain bitter-sensing, Gr66a-positive neurons but lack water cells. Expression of ppk28 in Gr66a-Gal4 neurons did not significantly affect the response to denatonium (G-CaMP imaging) or caffeine, endogenous ligands for Gr66a-Gal4 neurons. In response to water, Gr66a-Gal4 neurons showed no significant activity consistent with previous studies. Notably, misexpression of ppk28 in Gr66a-Gal4 neurons conferred sensitivity to water, as seen by extracellular bristle recordings and G-CaMP imaging. Moreover, the response was blunted as solute concentration was increased. Both NMDG and sucrose (substances that do not activate Gr66a-Gal4 neurons) produced dose-sensitive response decreases. The finding that both activation by water and inhibition by other compounds are conferred by ppk28 strongly suggests that ppk28 senses low osmolarity (Cameron, 2010).

To determine if ppk28 requires a taste cell environment to function or confers responsiveness to other cell-types, ppk28 was expressed in HEK293 heterologous cells. A FLAG-tagged ppk28 (inserted after amino acid 222 in the extracellular domain) was expressed in HEK293 cells, confirming that the protein was made and trafficked to the cell surface. For calcium imaging experiments, an untagged version of ppk28 was cotransfected with dsRed. Cells expressing the mammalian trpv4 osmo-sensitive ion channel were used as a positive control and cells transfected with the vector alone as a negative control. Cells were grown in a modified Ringers solution at 303 mmol/kg, loaded with Fluo-4 to visualize calcium changes and challenged with Ringers solution of different osmolalities. Cells transfected with vector alone showed a modest increase at 60% osmotic strength, whereas cells transfected with mammalian trpv4 showed fluorescence increases to all hypo-osmotic solutions, as expected. Importantly, cells transfected with ppk28 significantly responded to decreased osmolality, with dose-sensitive responses elicited by osmolalities of 216 and 174 mmol/kg. These experiments reveal that ppk28 bestows sensitivity to hypo-osmotic solutions in a variety of non-native environments and argue that the channel itself senses low osmolarity. This work provides a foundation for future studies of the biophysical properties of channel activation. Moreover, the ability to express ppk28 in heterologous cells and study its function creates the opportunity to compare its mechanism of gating with other Deg/ENaC family members involved in mechanosensation or sodium sensing (Cameron, 2010).

Overall, these studies examined the molecular basis for water taste detection in Drosophila and identified an ion channel belonging to the Deg/ENaC family, pickpocket 28 (ppk28), as the water gustatory sensor. This work demonstrates that an ion channel responding to low osmolarity mediates cellular and behavioral responses to water. Although the taste of water has received relatively little attention as a classic taste modality, water-responsive taste neurons have been described in many other insects, such as the blowfly and mosquitoes (Evans, 1962; Werner-Reiss, 1999), as well as in mammals, such as cats and rats (Lindemann, 1996; Gilbertson, 2002). The identification of ppk28 as a water taste receptor provides a framework for examining water taste detection in other animals, including humans (Cameron, 2010).

Osmosensation is important not only for the detection of external water sources by peripheral neurons but also for monitoring the plasma osmolality by central neurons. Several studies have identified members of the transient receptor potential family as candidate peripheral and central osmosensors, but the role of members of the Deg/ENaC family in osmosensation has received little attention. The finding that ppk28 is an osmosensitive ion channel raises the possibility that Deg/ENaC ion channels may participate broadly in peripheral and central osmosensation (Cameron, 2010).

The amiloride-sensitive epithelial Na+ channel PPK28 is essential for Drosophila gustatory water reception

Water sensation is a specific taste modality in the fruit fly. Water-induced hypoosmolarity activates specific gustatory receptor neurons; however, the molecular identity of the putative osmolarity sensor in these neurons remains unknown. This study found that amiloride and its analogs specifically antagonized the response of water gustatory receptor neurons and the behavior of flies toward water stimulation. Deletion of the gene that encodes the amiloride-sensitive PPK28 channel, a DEG/eNaC (degenerin/epithelial sodium channel) family member, abolished the water-induced activity of water gustatory receptor neurons and greatly diminished the behavioral response of flies to water. Ectopic expression of the PPK28 channel in the bitter cells within the intermediate-type sensilla renders these sensilla responsive to water stimuli. Thus, the amiloride-sensitive PPK28 channel may serve as the osmolarity sensor for gustatory water reception in the fruit fly (Chen, 2010).

Using pharmacological screening of a wide spectrum of ion channel antagonists, this study found that low concentrations of amiloride inhibited the spiking of water gustatory receptor neurons in Drosophila. Interestingly, several previous studies also demonstrated the effect of amiloride on the spiking of these receptor neurons in the flesh fly and blowfly (Liscia, 1997; Sadakata, 2002). Using physiological recordings, behavioral assays, and mutational analyses, this study identified PPK28 as the putative amiloride-sensitive receptor for gustatory water reception. Further gain-of-function studies showed that PPK28 is sufficient to confer hypoosmotic activity. These results suggest that PPK28 is a good candidate for the Drosophila gustatory water receptor (Chen, 2010).

The IC₅₀ of amiloride necessary to inhibit the gustatory water response (~20 μm) was about two orders of magnitude higher than that observed for mammalian ENaC; however, it was comparable to that used for the Drosophila RPK channel. It was found that the efficacy ranking of amiloride analogs in inhibiting gustatory water response was EIPA/DMA/MIA/HMA > DCB/amiloride >> CDPC. It is traditionally considered that EIPA/DMA/MIA/HMA are more specific in inhibiting Na⁺/Ca²⁺ or Na⁺/H⁺ exchangers compared with eNaC channels. Drosophila mutants for Na⁺/Ca²⁺ or Na⁺/H⁺ exchangers (Calx^A or Calx^B, Nhe2^f01515, and Nhe3^KG08307) retained robust water-elicited spiking in taste sensilla, which was in sharp contrast to that observed for the ppk28 deletion mutant. This result further supports the idea that PPK28 is the specific amiloride target involved in gustatory water reception (Chen, 2010).

While ppk28 deletion affected only water reception in both physiological and behavioral assays, it was found that higher concentrations of EIPA also suppressed other taste modalities. It is possible that other ppk channels and other amiloride targets, e.g., Na⁺/Ca²⁺ or Na⁺/H⁺ exchangers, are involved in modulating the reception and transduction of salt, sugar, or bitter signals in their respective receptor neurons. The identification of these EIPA targets in other GRNs may provide new insights into the understanding of other taste modalities. Although deletion of ppk28 resulted in a strong behavioral phenotype in water-induced PER responses, Δppk28 mutants displayed ~30% of PER response. This may be due to the following reasons. First, it is possible that the residual PER response may be contributed by taste-independent mechanisms, such as central regulatory mechanisms or other sensory signals (such as hygrosensation, vision, or mechanosensation), as the taste-deprived poxn^M22 mutants still displayed ~10% PER response to water under the assay conditions. In addition, although the water-elicited spikes from water cells can be almost completely abolished in Δppk28 mutants, sporadic spikes of small amplitude were recorded in Δppk28 mutant and wild-type flies. These small spikes were also seen in other work. Such small spikes could be activities from GRNs other than canonical water cells and could also contribute to the residual PER responses in Δppk28 mutants (Chen, 2010).

The mechanisms via which PPK28 is involved in Drosophila gustatory water reception remain unknown. The PPK28 channel itself may be mechanosensitive and gated in the same manner as that shown for several members of the DEG/eNaC channel family, e.g., MEC-4 in Caenorhabditis elegans, under the regulation of cytoskeleton or extracellular matrix proteins. Additional structure-function studies of PPK28 in Drosophila water GRNs may shed light on the molecular mechanisms that underlie osmosensation and mechanosensation in general (Chen, 2010).

Search PubMed for articles about Drosophila Ppk 28

Cameron, P., Hiroi, M., Ngai, J. and Scott, K. (2010). The molecular basis for water taste in Drosophila. Nature 465: 91-95. PubMed ID: 20364123

Chen, Z., Wang, Q. and Wang, Z. (2010). The amiloride-sensitive epithelial Na+ channel PPK28 is essential for Drosophila gustatory water reception. J Neurosci 30: 6247-6252. PubMed ID: 20445050

Evans, D. R. and Mellon, D., Jr. (1962). Electrophysiological studies of a water receptor associated with the taste sensilla of the blow-fly. J Gen Physiol 45: 487-500. PubMed ID: 13890971

Fischler, W., Kong, P., Marella, S. and Scott, K. (2007). The detection of carbonation by the Drosophila gustatory system. Nature 448: 1054-1057. PubMed ID: 17728758

Gilbertson, T. A. (2002). Hypoosmotic stimuli activate a chloride conductance in rat taste cells. Chem Senses 27: 383-394. PubMed ID: 12006378

Kellenberger, S. and Schild, L. (2002). Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol Rev 82: 735-767. PubMed ID: 12087134

Inoshita, T. and Tanimura, T. (2006). Cellular identification of water gustatory receptor neurons and their central projection pattern in Drosophila. Proc Natl Acad Sci U S A 103: 1094-1099. PubMed ID: 16415164

Liscia, A., Solari, P., Majone, R., Tomassini Barbarossa, I. and Crnjar, R. (1997). Taste reception mechanisms in the blowfly: evidence of amiloride-sensitive and insensitive receptor sites. Physiol Behav 62: 875-879. PubMed ID: 9284511

Lindemann, B. (1996). Taste reception. Physiol Rev 76: 718-766. PubMed ID: 8757787

Meunier, N., Ferveur, J. F. and Marion-Poll, F. (2000). Sex-specific non-pheromonal taste receptors in Drosophila. Curr Biol 10: 1583-1586. PubMed ID: 11137009

Meunier, N., Marion-Poll, F. and Lucas, P. (2009). Water taste transduction pathway is calcium dependent in Drosophila. Chem Senses 34: 441-449. PubMed ID: 19386695

Sadakata, T., Hatano, H., Koseki, T., Koganezawa, M. and Shimada, I. (2002). The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina. J Insect Physiol 48: 565-570. PubMed ID: 12770084

Werner-Reiss, U., Galun, R., Crnjar, R. and Liscia, A. (1999). Sensitivity of the mosquito Aedes aegypti (Culicidae) labral apical chemoreceptors to blood plasma components. J Insect Physiol 45: 485-491. PubMed ID: 12770332

Yarmolinsky, D. A., Zuker, C. S. and Ryba, N. J. (2009). Common sense about taste: from mammals to insects. Cell 139: 234-244. PubMed ID: 19837029

date revised: 15 February 2013

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