CALHM3 Is Essential for Rapid Ion Channel-Mediated Purinergic Neurotransmission of GPCR-Mediated Tastes

Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes. Ma et al. identify a CALHM1/CALHM3 hetero-hexameric ion channel as the mechanism by which type II taste bud cells release ATP as a neurotransmitter to gustatory neurons in response to GPCR-mediated tastes, including sweet, bitter, and umami substances. © 2018 Elsevier Inc

Modulation of sweet taste sensitivities by endogenous leptin and endocannabinoids in mice.

Key pointsPotential roles of endogenous leptin and endocannabinoids in sweet taste were examined by using pharmacological antagonists and mouse models including leptin receptor deficient (db/db) and diet-induced obese (DIO) mice. Chorda tympani (CT) nerve responses of lean mice to sweet compounds were increased after administration of leptin antagonist (LA) but not affected by administration of cannabinoid receptor antagonist (AM251). db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid levels in the taste organ, and enhanced expression of a biosynthesizing enzyme of endocannabinoids in taste cells. The effect of LA was gradually decreased and that of AM251 was increased during the course of obesity in DIO mice. These findings suggest that circulating leptin, but not local endocannabinoids, is a dominant modulator for sweet taste in lean mice and endocannabinoids become more effective modulators of sweet taste under conditions of deficient leptin signalling.AbstractLeptin is an anorexigenic mediator that reduces food intake by acting on hypothalamic receptor Ob-Rb. In contrast, endocannabinoids are orexigenic mediators that act via cannabinoid CB1 receptors in hypothalamus, limbic forebrain, and brainstem. In the peripheral taste system, leptin administration selectively inhibits behavioural, taste nerve and taste cell responses to sweet compounds. Opposing the action of leptin, endocannabinoids enhance sweet taste responses. However, potential roles of endogenous leptin and endocannabinoids in sweet taste remain unclear. Here, we used pharmacological antagonists (Ob-Rb: L39A/D40A/F41A (LA), CB1 : AM251) and examined the effects of their blocking activation of endogenous leptin and endocannabinoid signalling on taste responses in lean control, leptin receptor deficient db/db, and diet-induced obese (DIO) mice. Lean mice exhibited significant increases in chorda tympani (CT) nerve responses to sweet compounds after LA administration, while they showed no significant changes in CT responses after AM251. In contrast, db/db mice showed clear suppression of CT responses to sweet compounds after AM251, increased endocannabinoid (2-arachidonoyl-sn-glycerol (2-AG)) levels in the taste organ, and enhanced expression of a biosynthesizing enzyme (diacylglycerol lipase α (DAGLα)) of 2-AG in taste cells. In DIO mice, the LA effect was gradually decreased and the AM251 effect was increased during the course of obesity. Taken together, our results suggest that circulating leptin, but not local endocannabinoids, may be a dominant modulator for sweet taste in lean mice; however, endocannabinoids may become more effective modulators of sweet taste under conditions of deficient leptin signalling, possibly due to increased production of endocannabinoids in taste tissue

<i>Gli3</i> is a negative regulator of <i>Tas1r3</i>-expressing taste cells

<div><p>Mouse taste receptor cells survive from 3–24 days, necessitating their regeneration throughout adulthood. In anterior tongue, sonic hedgehog (SHH), released by a subpopulation of basal taste cells, regulates transcription factors <i>Gli2</i> and <i>Gli3</i> in stem cells to control taste cell regeneration. Using single-cell RNA-Seq we found that <i>Gli3</i> is highly expressed in <i>Tas1r3</i>-expressing taste receptor cells and <i>Lgr5+</i> taste stem cells in posterior tongue. By PCR and immunohistochemistry we found that <i>Gli3</i> was expressed in taste buds in all taste fields. Conditional knockout mice lacking <i>Gli3</i> in the posterior tongue (<i>Gli3</i>^<i>CKO</i>) had larger taste buds containing more taste cells than did control wild-type (<i>Gli3</i>^<i>WT</i>) mice. In comparison to wild-type mice, <i>Gli3</i>^<i>CKO</i> mice had more <i>Lgr5</i>+ and <i>Tas1r3</i>+ cells, but fewer type III cells. Similar changes were observed <i>ex vivo</i> in <i>Gli3</i>^<i>CKO</i> taste organoids cultured from <i>Lgr5+</i> taste stem cells. Further, the expression of several taste marker and <i>Gli3</i> target genes was altered in <i>Gli3</i>^<i>CKO</i> mice and/or organoids. Mirroring these changes, <i>Gli3</i>^<i>CKO</i> mice had increased lick responses to sweet and umami stimuli, decreased lick responses to bitter and sour taste stimuli, and increased glossopharyngeal taste nerve responses to sweet and bitter compounds. Our results indicate that <i>Gli3</i> is a suppressor of stem cell proliferation that affects the number and function of mature taste cells, especially <i>Tas1r3</i>+ cells, in adult posterior tongue. Our findings shed light on the role of the Shh pathway in adult taste cell regeneration and may help devise strategies for treating taste distortions from chemotherapy and aging.</p></div

<i>Gli3</i>-deficient mice exhibit increased behavioral responses to sweet, umami, and bitter tastants.

<p>Brief-access tests were used to measure behavioral responses to sweet (sucrose and sucralose, A and B), umami (monosodium glutamate [MSG], C), bitter (denatonium, D), salty (NaCl, E), and sour (citric acid, F) taste stimuli. Compared to <i>Gli3</i>^<i>WT</i> mice, <i>Gli3</i>^<i>CKO</i> mice display increased lick responses over a range of concentrations to the appetitive stimuli (sucrose, sucralose, and MSG) and decreased lick responses to the aversive stimuli (denatonium and citric acid). Lick ratios were calculated by dividing the number of licks to a taste solution by the number of licks to water in each test session. Data are means ± SEM Statistically significant differences were determined by repeated two-way ANOVA test [sucrose: F (1, 119) = 13.07, P<0.05; sucralose: F (1, 159) = 9.76, P<0.05; MSG: F (1, 119) = 8.69, P<0.05; denatonium: F (1, 123) = 5.40, P<0.05; NaCl: F (1, 147) = 1.57, P>0.05; citric acid: F (1, 127) = 5.45, P<0.05] and post hoc t-test (n>12, *<i>p</i><0.05, **<i>p</i><0.01).</p

Effect of <i>Gli3</i> deficiency on taste bud size and composition.

<p>(A-H) Indirect immunofluorescence confocal microscopy of CV sections from (<i>Gli3</i>^<i>WT</i>) <i>Gli3</i> conditional knockout (<i>Gli3</i>^<i>CKO</i>) mice immunostained for all taste cells with antibodies against KCNQ1 (A, B) and immunostained for type II taste cells using markers TRPM5 (C, D), T1R3 (E, F), and GNAT3 (G, H). Nuclei are counterstained with DAPI (blue). Scale bars, 100 μm. (I) Compared to control (<i>Gli3</i>^<i>WT</i>) mice, the number of taste buds did not change in <i>Gli3</i>^<i>CKO</i> mice (t = 1.53, p>0.05), but the size (in μm²) (t = 2.26, p<0.05) and the number of taste cells (t = 4.58, p<0.001) in taste buds increased. (J) Compared to <i>Gli3</i>^<i>WT</i> mice, the density of TRPM5+ (t = 9.44, p<0.0001) and T1R3+ (t = 8.94, p<0.0001) but not GNAT3+ (t = 1.49, p>0.05) taste receptor cells in <i>Gli3</i>^<i>CKO</i> mice increased. (K) qPCR showed that Trpm5 (t = 6.67, p<0.01), Tas1r3 (t = 12.28, p<0.001) and Gna14 (t = 13.42, p<0.0001) but not Gnat3 (t = 0.87, p>0.05) mRNA expression increased in CV taste buds from <i>Gli3</i>^<i>CKO</i> mice relative to those of <i>Gli3</i>^<i>WT</i> mice. Five control and <i>Gli3</i>^<i>CKO</i> mice each were used for analyses. Data are means + SEM. *<i>p</i><0.05 **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001.</p

GLI3 is selectively expressed in T1R3+ and TRPM5+ type II taste receptor cells.

<p>Double-labeled indirect immunofluorescence confocal microscopy of sections from mouse fungiform papilla (FF; A-C), folate papillae (FO; D-F), and circumvallate (CV; G-I) papillae was performed with antibodies against GLI3 and TRPM5 (B, E, H) or intrinsic GFP fluorescence in <i>Tas1r3</i>-GFP (A, D, G) and <i>Gnat3</i>-GFP (C, F, I) transgenic mice. Overlaid images show frequent co-expression of GLI3 with <i>Tas1r3</i>-GFP and TRPM5 but not with <i>Gnat3</i>-GFP. Scale bars, 50 μm.</p

<i>Gli3</i> deficiency affects taste cell differentiation and expression of Shh target genes.

<p>(A-D) Indirect immunofluorescence confocal microscopy of taste organoids cultured from individual FACS-sorted Lgr5-GFP cells isolated from CV papillae of double-knockin mice treated with tamoxifen (<i>Gli3</i>^<i>CKO</i>; C, D) or without (<i>Gli3</i>^<i>WT</i>;A, B) and stained with antibodies against T1R3 (A, C) or GNAT3 (B, D). Scale bars, 100 μm. (E) The number of T1R3+ (n = 76, t = 3.05, p<0.01) but not GNAT3+ (n = 84, t = 0.61, p>0.05) cells increased in <i>Gli3</i>^<i>CKO</i> organoids. (F) In <i>Gli3</i>^<i>CKO</i> organoids vs. those from <i>Gli3</i>^<i>WT</i> the expression of <i>Tas1r3</i> mRNA (t = 8.87, p<0.001) increased dramatically, while that of <i>Gna14(t =</i>, <i>p<0</i>.<i>01)</i> and <i>Gnat3</i> (t = 4.37, p<0.05) showed a modest increase, but that of <i>Trpm5 (t = 1</i>.<i>17</i>, <i>p>0</i>.<i>05)</i> remained unchanged. Data are means + SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Increased taste nerve responses to sweet and bitter stimuli in <i>Gli3</i>^<i>CKO</i> mice.

<p>Average glossopharyngeal (GL) nerve responses to sweet (sucrose and sucralose, A and B), umami (monosodium glutamate [MSG], C), bitter (denatonium, D), salty (NaCl, E), and sour (citric acid, F) tasting compounds are shown. The magnitude of the responses to taste compounds was normalized against the response to 100 mM NH₄Cl. Compared to <i>Gli3</i>^<i>WT</i> mice, <i>Gli3</i>^<i>CKO</i> mice exhibit increased nerve responses to sucrose, sucralose, and denatonium across a range of concentrations, while the responses to other stimuli were unaffected. Statistically significant differences were determined by repeated two-way ANOVA test [sucrose: F (1, 63) = 12.32, P<0.05; sucralose: F (1, 78) = 9.67, P<0.05; MSG: F (1, 71) = 0.71, P>0.05; denatonium: F (1, 68) = 14.81, P<0.05; NaCl: F (1, 67) = 0.36, P>0.05; citric acid: F(1, 72) = 1.51, P>0.05] and post hoc t-test (n≥7, *p<0.01). All data are presented as the means±SEM.</p