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The molecular basis for water taste in Drosophila.

Cameron P, Hiroi M, Ngai J, Scott K - Nature (2010)

Bottom Line: Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water.We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that loss of ppk28 abolishes water sensitivity.These studies link an osmosensitive ion channel to water taste detection and drinking behaviour, providing the framework for examining the molecular basis for water detection in other animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, CA, USA.

ABSTRACT
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, but little is known about the underlying molecular mechanism for water taste detection. Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water. We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that 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 behaviour, providing the framework for examining the molecular basis for water detection in other animals.

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The ppk28 gene is necessary for cellular and behavioral water responses. a. Extracellular bristle recordings of ppk28 control, mutant and rescue flies after water (left) or 100 mM sucrose (right) stimulation, showing action potentials. Stimulation begins at recording. b. Scatter plot of water and sugar responses (mean ± s.e.m in bars; data points as dots). Water responses are ***P=0.001 by Dunn's multiple comparison. c. G-CaMP fluorescence increase in ppk28 control, mutant and rescue projections to water (%maxΔF/F) (SOG, scale bar 50μm). d. Fluorescence change summary following water, 0.1M NaCl, 1M NaCl, 1M sucrose (n=8-11 trials/concentration ± s.e.m; t-test, ppk28 control versus mutant, water: ***P=0.0008, 1M NaCl: *P=0.03). e. Behavioral assays measuring water or 500mM sucrose consumption time. Control flies drink more water than ppk28 mutants (*P=0.017), ppk28 mutants + ppk28-Gal4 (*P=0.037) or ppk28 mutants + UAS-ppk28 (**P=0.008). Water consumption of control and rescue is not different (P=0.53). Sucrose consumption is not different (vs control, mutant: P=0.63; rescue: P=0.53). n= 3 ± s.e.m trials, 18-25 flies/trial/genotype, t-test.
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Figure 2: The ppk28 gene is necessary for cellular and behavioral water responses. a. Extracellular bristle recordings of ppk28 control, mutant and rescue flies after water (left) or 100 mM sucrose (right) stimulation, showing action potentials. Stimulation begins at recording. b. Scatter plot of water and sugar responses (mean ± s.e.m in bars; data points as dots). Water responses are ***P=0.001 by Dunn's multiple comparison. c. G-CaMP fluorescence increase in ppk28 control, mutant and rescue projections to water (%maxΔF/F) (SOG, scale bar 50μm). d. Fluorescence change summary following water, 0.1M NaCl, 1M NaCl, 1M sucrose (n=8-11 trials/concentration ± s.e.m; t-test, ppk28 control versus mutant, water: ***P=0.0008, 1M NaCl: *P=0.03). e. Behavioral assays measuring water or 500mM sucrose consumption time. Control flies drink more water than ppk28 mutants (*P=0.017), ppk28 mutants + ppk28-Gal4 (*P=0.037) or ppk28 mutants + UAS-ppk28 (**P=0.008). Water consumption of control and rescue is not different (P=0.53). Sucrose consumption is not different (vs control, mutant: P=0.63; rescue: P=0.53). n= 3 ± s.e.m trials, 18-25 flies/trial/genotype, t-test.

Mentions: To determine the function of ppk28 in the water response, we generated a ppk28 mutant by piggybac transposon mediated gene deletion, removing 1.769kb surrounding the ppk28 gene16. We examined the water responses of ppk28 control, mutant and rescue flies 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 cells3. Control flies showed 12.0±0.9 spikes/sec when stimulated with water (Fig. 2a, b). 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 (Fig. 2a, b). Responses to sucrose were not significantly different among the three genotypes (58.9±3.3 spikes/sec, 46.9±2.6 spikes/sec and 49.0±1.8 spikes/sec, for control, mutant and rescue flies, respectively) (Fig. 2a, b), 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 (Fig. 2c, d). Taken together, the electrophysiological and imaging data demonstrate that ppk28 is required for the cellular response to water.


The molecular basis for water taste in Drosophila.

Cameron P, Hiroi M, Ngai J, Scott K - Nature (2010)

The ppk28 gene is necessary for cellular and behavioral water responses. a. Extracellular bristle recordings of ppk28 control, mutant and rescue flies after water (left) or 100 mM sucrose (right) stimulation, showing action potentials. Stimulation begins at recording. b. Scatter plot of water and sugar responses (mean ± s.e.m in bars; data points as dots). Water responses are ***P=0.001 by Dunn's multiple comparison. c. G-CaMP fluorescence increase in ppk28 control, mutant and rescue projections to water (%maxΔF/F) (SOG, scale bar 50μm). d. Fluorescence change summary following water, 0.1M NaCl, 1M NaCl, 1M sucrose (n=8-11 trials/concentration ± s.e.m; t-test, ppk28 control versus mutant, water: ***P=0.0008, 1M NaCl: *P=0.03). e. Behavioral assays measuring water or 500mM sucrose consumption time. Control flies drink more water than ppk28 mutants (*P=0.017), ppk28 mutants + ppk28-Gal4 (*P=0.037) or ppk28 mutants + UAS-ppk28 (**P=0.008). Water consumption of control and rescue is not different (P=0.53). Sucrose consumption is not different (vs control, mutant: P=0.63; rescue: P=0.53). n= 3 ± s.e.m trials, 18-25 flies/trial/genotype, t-test.
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Figure 2: The ppk28 gene is necessary for cellular and behavioral water responses. a. Extracellular bristle recordings of ppk28 control, mutant and rescue flies after water (left) or 100 mM sucrose (right) stimulation, showing action potentials. Stimulation begins at recording. b. Scatter plot of water and sugar responses (mean ± s.e.m in bars; data points as dots). Water responses are ***P=0.001 by Dunn's multiple comparison. c. G-CaMP fluorescence increase in ppk28 control, mutant and rescue projections to water (%maxΔF/F) (SOG, scale bar 50μm). d. Fluorescence change summary following water, 0.1M NaCl, 1M NaCl, 1M sucrose (n=8-11 trials/concentration ± s.e.m; t-test, ppk28 control versus mutant, water: ***P=0.0008, 1M NaCl: *P=0.03). e. Behavioral assays measuring water or 500mM sucrose consumption time. Control flies drink more water than ppk28 mutants (*P=0.017), ppk28 mutants + ppk28-Gal4 (*P=0.037) or ppk28 mutants + UAS-ppk28 (**P=0.008). Water consumption of control and rescue is not different (P=0.53). Sucrose consumption is not different (vs control, mutant: P=0.63; rescue: P=0.53). n= 3 ± s.e.m trials, 18-25 flies/trial/genotype, t-test.
Mentions: To determine the function of ppk28 in the water response, we generated a ppk28 mutant by piggybac transposon mediated gene deletion, removing 1.769kb surrounding the ppk28 gene16. We examined the water responses of ppk28 control, mutant and rescue flies 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 cells3. Control flies showed 12.0±0.9 spikes/sec when stimulated with water (Fig. 2a, b). 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 (Fig. 2a, b). Responses to sucrose were not significantly different among the three genotypes (58.9±3.3 spikes/sec, 46.9±2.6 spikes/sec and 49.0±1.8 spikes/sec, for control, mutant and rescue flies, respectively) (Fig. 2a, b), 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 (Fig. 2c, d). Taken together, the electrophysiological and imaging data demonstrate that ppk28 is required for the cellular response to water.

Bottom Line: Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water.We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that loss of ppk28 abolishes water sensitivity.These studies link an osmosensitive ion channel to water taste detection and drinking behaviour, providing the framework for examining the molecular basis for water detection in other animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, CA, USA.

ABSTRACT
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, but little is known about the underlying molecular mechanism for water taste detection. Here we identify a member of the degenerin/epithelial sodium channel family, PPK28, as an osmosensitive ion channel that mediates the cellular and behavioural response to water. We use molecular, cellular, calcium imaging and electrophysiological approaches to show that ppk28 is expressed in water-sensing neurons, and that 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 behaviour, providing the framework for examining the molecular basis for water detection in other animals.

Show MeSH
Related in: MedlinePlus