Alkaline taste sensation through the alkaliphile chloride channel in Drosophila

Jun 18, 2023

Nature Metabolism volume 5, pages 466–480 (2023)Cite this article

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The sense of taste is an important sentinel governing what should or should not be ingested by an animal, with high pH sensation playing a critical role in food selection. Here we explore the molecular identities of taste receptors detecting the basic pH of food using Drosophila melanogaster as a model. We identify a chloride channel named alkaliphile (Alka), which is both necessary and sufficient for aversive taste responses to basic food. Alka forms a high-pH-gated chloride channel and is specifically expressed in a subset of gustatory receptor neurons (GRNs). Optogenetic activation of alka-expressing GRNs is sufficient to suppress attractive feeding responses to sucrose. Conversely, inactivation of these GRNs causes severe impairments in the aversion to high pH. Altogether, our discovery of Alka as an alkaline taste receptor lays the groundwork for future research on alkaline taste sensation in other animals.

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All relevant data have been presented in this paper and its supplementary information. Any other related information is available upon request from Y.V.Z. In addition, we deposited the raw confocal videos for double-labeling experiments, including Fig. 3h (https://doi.org/10.6084/m9.figshare.22029284), Fig. 3i (https://doi.org/10.6084/m9.figshare.22029131), Fig. 3k (https://doi.org/10.6084/m9.figshare.22029488) and Fig. 3l (https://doi.org/10.6084/m9.figshare.22029350) in figshare, a publicly accessible repository. Source data are provided with this paper.

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We thank S. Chan and P. Nguyen for their technical contributions to this study. We also thank the Bloomington Drosophila Research Center for fly stocks and the Drosophila Genome Research Center for DNA clones. We appreciate the laboratories of D.R. Reed, M. Hakan Ozdener and I. Matsumoto at the Monell Chemical Senses Center for sharing equipment and facilities. Our work was supported by the National Institute on Deafness and other Communication Disorders grants R01 DC018592 (Y.V.Z.) and R01 DC007864 (C.M.), the Ambrose Monell Foundation (Y.V.Z.) and the National Key Research and Development Program of China Project 2018YFA0108001 (Z.-Q.T.).

These authors contributed equally: Tingwei Mi, John O. Mack.

Monell Chemical Senses Center, Philadelphia, PA, USA

Tingwei Mi, John O. Mack, Wyatt Koolmees, Quinn Lyon, Luke Yochimowitz, Peihua Jiang & Yali V. Zhang

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China

Zhao-Qian Teng

Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA

Craig Montell

Department of Physiology, The Diabetes Research Center, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

Yali V. Zhang

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Y.V.Z. conceived of this work. T.M., J.O.M., W.K., Q.L. and L.Y. carried out the molecular genetics and feeding experiments. T.M. and Z.-Q.T. performed patch-clamp analyses. W.K., P.J., L.Y. and Y.V.Z. conducted immunohistochemistry and tip-recording assays. T.M., J.O.M., W.K., C.M. and Y.V.Z. interpreted the data and composed the paper. Y.V.Z. oversaw the project.

Correspondence to Yali V. Zhang.

The authors declare no competing interests.

Nature Metabolism thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Ashley Castellanos-Jankiewicz, in collaboration with the Nature Metabolism team.

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a, We tested a wide array of receptor and ion channel candidates, a representative sample of which is shown in the bar graph. These include the gustatory receptor (Gr) family, such as Gr66a and Gr33a; the ionotropic glutamate receptor (Ir) family, such as Ir76b and Ir25a; the transient-receptor-potential (trp) ion channel family, such as trpl and trpA1; the otopetrin family, such as otopla; the transmembrane channel-like (tmc); and genes with unknown functions, such as CG12344. n = 10 trials. b, Feeding responses to neutral versus alkaline foods among control flies and mutant flies of the fly LGCC family. n = 10 trials. Mean ± s.e.m., one-way ANOVA with Tukey's posthoc tests, ****p < 0.0001.

Source data

a, Genomic composition of the wild-type and mutant alka genes, including the translational start (ATG) and stop (*) codons, exons, and introns. The red arrows indicate the guide RNA (gRNA) target sites (gRNA1 and gRNA2). To screen for the alka1 mutant, we designed three sets of primers, P1, P2, and P3, which flanked the gRNA targeting sites. b, PCR analyses of genomic DNA with the P1, P2, and P3 primers for wild-type (wt) and alka1 mutant flies. c, The predicted topology of the Alka protein comprising four transmembrane segments. The TM2 domain (red) is predicted to line the channel pore. Both the N- and C-terminal ends of the Alka protein reside on the extracellular side. The red arrows show the ablated protein regions in the alka1 mutant.

a, Statistical analyses of the frequencies of spikes produced by L-, I-, and S-type sensilla responding to 10 mM NaOH in wild-type (wt) flies. n = 11 flies. Mean ± s.e.m., one-way ANOVA with Tukey's posthoc tests, ****p < 0.0001. b, Representative spikes evoked by 10 mM NaCl (pH 7) at S6 sensilla for wt and alka1 mutant flies. The arrow indicates the stimulus onset. c, Statistical analyses of the frequencies of spikes produced by S6 sensilla in response to different concentrations of NaCl for wt and alka1 mutant flies. n = 10 flies. Mean ± s.e.m., unpaired two-tailed Student's t-tests.

Source data

a–b, Expression of alka-gal4;UAS-mCD8::GFP at the tarsal segment of the fly foreleg. c, PERs to alkaline solutions containing 30 mM sucrose and various concentrations of NaOH among wild-type (wt), alka1, alka1;Ir761, and rescue flies. n = 12 trials. Mean ± s.e.m., two-way ANOVA with Tukey's posthoc tests, **p = 0.0081, ****p < 0.0001. d, Expression of alka-gal4;UAS-mCD8::GFP in the maxillary palp. e, Expression of alka-gal4;UAS-mCD8::GFP in the antenna. f, No obvious anti-Alka signals were detected in the wt adult brain. g, Relative localization pattern between ppk28-expressing GRNs and taste sensilla. Scale bars: 10 μm (a–b, d–e, g), 50 μm (f).

Source data

The protein sequence identities between Alka and GlyRa1 in other species are as follows: zebrafish, 30% identity; mouse, 30% identity; human, 30% identity. Identical amino acid residues are labeled in red, whereas similar amino acid residues among at least three species are labeled in yellow and highlighted in bold. The four transmembrane regions (TM1-TM4) are denoted by black bars above their amino acid sequences.

a, Localization of Alka in HEK293 cells expressing Alka that is N-terminally fused with a Myc tag. Scale bar: 5 μm. b, Configuration of the whole-cell patch-clamp recording setup. We used a stimulating pipette (red) to locally apply high-pH solutions to the cells and a patch pipette (blue) to carry out whole-cell recordings. c, Current-voltage (I-V) relationships of Alka-expressed HEK293 cells, which were elicited by voltage ramps from -80 mV to +80 mV. The bath solution contained 150 mM NaF, NaCl, NaBr, or NaI, and the intracellular solution contained 150 mM CsCl. d, Statistical analyses of reversal potentials from experiments in c. n = 11 cells (NaF, NaBr, or NaI); n = 14 cells (NaCl). Mean ± s.e.m., one-way ANOVA with Tukey's posthoc tests, **p = 0.0012, ****p < 0.0001. e, Relative anion permeability of Alka. n = 11 cells (NaF, NaBr, or NaI); n = 14 cells (NaCl). Mean ± s.e.m., one-way ANOVA with Tukey's posthoc tests, ***p = 0.0003. f, g, Currents from Alka-expressing cells and control cells without Alka expression responding to the stimuli of acidic isosmotic solutions. n = 11 cells. Mean ± s.e.m., unpaired two-tailed Student's t-tests. h–k, Currents evoked by Alka-expressing cells and control cells without Alka expression in response to the stimuli of glycine (0.001–1 mM) (h,i) or GABA (0.001–1 mM) (j,k). n = 11 cells. Mean ± s.e.m., unpaired two-tailed Student's t-tests. Cells were clamped at −70 mV. Arrows indicate the onset of stimulus.

Source data

a, Spikes evoked by L7 sensilla responding to high-pH stimuli in alka1;Gr64f-Gal4 or alka1;UAS-alka flies. b, Statistical analyses of the spike frequencies for alka1;UAS-alka and alka1;Gr64f-Gal4 flies. n = 11 flies. Mean ± s.e.m., unpaired two-tailed Student's t-tests. c, Spikes produced by L7 sensilla responding to 50 mM sucrose in alka1;Gr64f-Gal4/UAS-alka or alka1;Gr64f-Gal4/UAS-alkaP276A flies. d, Statistical analyses of the spike frequencies for alka1;Gr64f-Gal4/UAS-alka and alka1;Gr64f-Gal4/UAS-alkaP276A flies. n = 11 flies. Mean ± s.e.m., unpaired two-tailed Student's t-tests. Arrows indicate stimulus onset.

Source data

a–b, Expression of alka-Gal4,UAS-mCD8::GFP;Orco-Gal80 in the antenna (a) and the maxillary palp (b). c, Expression of alka-Gal4,UAS-mCD8::GFP;Orco-Gal80 in the labellum. d, GRN projections in the brain of the alka-Gal4,UAS-mCD8::GFP;Orco-Gal80 fly. SEZ, subesophageal zone; AL, antennal lobe. Scale bars: 10 μm (a–c), 50 μm (d).

a–b, GFP expression in the labellum of Gr66a-Gal4,UAS-GFP;LexAop-Gal80 (a) or Gr66a-Gal4,UAS-GFP;Gr66a-lexA,LexAop-Gal80 (b). Scale bar: 10 μm. c, Relative localization between the alka-expressing GRNs and S-type taste sensilla in the alka-Gal4,UAS-GFP;Gr66a-lexA,LexAop-Gal80 fly labellum. Scale bar: 10 μm. d, PERs to sweet food (50 mM sucrose), alkaline food (10 mM NaOH mixed with 30 mM sucrose), and bitter food (10 mM caffeine mixed with 30 mM sucrose) for alka-TNT (alka-Gal4,UAS-TNT), alka-TNT;Orco-Gal80 (alka-Gal4,UAS-TNT;Orco-Gal80), alka-TNT;Gr66a-Gal80(alka-Gal4,UAS-TNT;Gr66a-lexA,LexAop-Gal80), Gr66a-TNT(Gr66a-Gal4,UAS-TNT),Gr66a-TNT;Gr66a-Gal80 (Gr66a-Gal4,UAS-TNT;Gr66a-lexA,LexAop-Gal80), ppk23-TNT (ppk23-Gal4,UAS-TNT), ppk28-TNT (ppk28-Gal4,UAS-TNT), and wild-type (wt) flies. n = 11 trials. Mean ± s.e.m., one-way ANOVA with Tukey's posthoc tests, ****p < 0.0001.

Source data

Supplementary Sections 1 (Antibodies), 2 (Molecular genetics) and 3 (Extended Data video titles).

The alka-Gal4 control fly showing persistent sucrose (500 mM) feeding in the presence and absence of an intense red light stimulus (2,000 lux).

The alka-Gal4,UAS-CsChrimson;Orco-Gal80 fly exhibiting normal feeding of sucrose (500 mM) in the absence of red light but a cessation of feeding in response to a moderate red light stimulus (1,200 lux).

Statistical source data.

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Mi, T., Mack, J.O., Koolmees, W. et al. Alkaline taste sensation through the alkaliphile chloride channel in Drosophila. Nat Metab 5, 466–480 (2023). https://doi.org/10.1038/s42255-023-00765-3

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Received: 15 August 2022

Accepted: 09 February 2023

Published: 20 March 2023

Issue Date: March 2023

DOI: https://doi.org/10.1038/s42255-023-00765-3

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