Salt taste sensation.

Approximate concentration ranges of salt taste perceptions in humans (Bartoshuk et al., 1978) and qualities of taste sensation with known cells and receptors responsible for their sensing are summarized.

Cl−-binding sites in the medaka fish taste receptor T1r2a/T1r3LBD.

Schematic drawing of the overall architecture of T1r2a/T1r3. The crystal structure (PDB ID: 5X2M) (Nuemket et al., 2017) is shown at the LBD region, and helices B and C in T1r3 are labeled. (B) Anomalous difference Fourier map (4.5 σ, red) of the Br−-substituted T1r2a/T1r3LBD crystal. (C) Anomalous difference Fourier map (4.5 σ, red) of the Cl -bound T1r2a/T1r3LBD crystal derived from the diffraction data collected at the wavelength of 2.7 Å. In panels B and C, the site originally identified the Cl−-binding was framed. (D) A close-up view of the Cl−-binding site in T1r3LBD in the Cl−-bound T1r2a/T1r3LBD (PDB ID: 5X2M). (E) Amino-acid sequence alignment of T1r proteins and the related receptors at the Cl−-binding site. The “h,” “m,” and “mf” prefixes to T1rs indicate human, mouse, and medaka fish, respectively. The position corresponding to Thr105 in T1r3 from medaka fish is highlighted. (F) The structures of ANPR (PDB ID: 1T34, left) (Ogawa et al, 2004) and mGluR2 (PDB ID: 5CNI, right) (Monn et al, 2015a) bound with Cl−. (G) Superposition of the Cl−-binding site in T1r3, ANPR, and mGluR2.

The Cl−-binding properties of T1r2a/T1r3LBD.

(A) Representative thermal melt curves of T1r2a/T1r3LBD in the presence of 0.003 –10 mM concentrations of Cl− measured using DSF. (B) Dose-dependent Tm changes of T1r2a/T1r3LBD by addition of Cl−. (n = 4) (C) Thermal melting curves of WT and the T1r3-T105A mutant of T1r2a/T1r3LBD in the presence and absence of Cl−, analyzed by FSEC-TS. (n = 1) (D) Dose-dependent FRET signal changes of the T1r2aLBD-Cerulean and T1r3LBD-Venus heterodimer by addition of Cl−. (n = 3) (E) FRET index increases by adding 10-mM Cl− or 1-mM L-glutamine to the WT or T1r3-T105A mutant T1r2aLBD-Cerulean/T1r3LBD-Venus heterodimer relative to that in the absence of any ligand in the absence of Cl−. (n = 3) (F) Dose-dependent FRET signal changes of the T1r2aLBD-Cerulean and T1r3LBD-Venus heterodimer induced by the addition of L-glutamine in the presence and absence of Cl−. (n = 3) The experiments were performed two (panels A, B, D, and E), three (C and WT), four (F and +Cl− condition), or one (C and mutant; F and –Cl− condition) time(s), and the results from one representative experiment are shown with numbers of technical replicates. Data points represent mean and s.e.m.

Properties of the Cl−-binding to T1r2a/T1r3LBD.

Electrophysiological and behavioral analyses of the T1r-mediated Cl− responses in mouse.

(A-D) Results of single fiber recordings from the mouse chorda tympani nerve. (A) Representative recordings of single fibers that connect to T1r-expressing taste cells. The stimuli were 10-mM NMDG-Cl, 100-mM sucrose, 100-mM sucrose + 10-mM NMDG-Cl, 20-mM L-glutamine, or 20-mM L-glutamine + 10-mM NMDG-Cl. Lines indicate the application of stimuli to the tongue. All the responses were suppressed by lingual treatment with a T1r blocker, Gur (right). (B) Impulse frequencies in response to the concentration series of NMDG-Cl, NaCl, or KCl before and after Gur treatment in WT mice. Responses to NMDG-gluconate are also shown. The mean number of net impulses per 10 s (mean response) ± s.e.m. in Gur-sensitive fibers (n = 5–6 from six mice). (C) Impulse frequencies in response to the concentration series of NMDG-Cl before and after Gur treatment were measured in T1r3-KO mice (n = 4-5 from three mice). Responses to NMDG-gluconate are also shown. (D) Impulse frequencies to 20-mM L-glutamine or 100-mM sucrose in the absence or presence of 10-mM NMDG-Cl before and after Gur treatment. Responses to 20-mM L-glutamine or 100-mM sucrose by T1r3-KO mouse are also shown. Values are mean ± s.e.m. (n = 3–5 from three mice each). *, **: paired t-test; P < 0.05 (*) and < 0.01 (**). (E) Amount of fluid intake for water and 10-mM NMDG-Cl in the two-bottle preference tests. Values are mean ± s.e.m. (n = nine mice) (F) NMDG-Cl intake shown in (E) normalized to water intake (preference score) in the two-bottle preference tests. A score > 50% indicates that the taste solution was preferred over water.