Spilanthol Enhances Sensitivity to Sodium in Mouse Taste Bud Cells

Abstract

Overconsumption of NaCl has been linked to increased hypertension-related morbidity. Compounds that can enhance NaCl responses in taste cells could help reduce human NaCl consumption without sacrificing perceived saltiness. Spilanthol is an unsaturated alkylamide isolated from the Jambu plant (Acmella oleracea) that can induce tingling, pungency, and numbing in the mouth. Structurally similar fatty acid amides, such as sanshool, elicit numbing and tingling sensations by inhibiting 2-pore-domain potassium leak channels on trigeminal sensory neurons. Even when insufficient to induce action potential firing, leak current inhibition causes depolarization and increased membrane resistance, which combine to make cells more sensitive to subsequent depolarizing stimuli, such as NaCl. Using calcium imaging, we tested whether spilanthol alters sensitivity to NaCl in isolated circumvallate taste bud cells and trigeminal sensory neurons of mice (Mus musculus). Micromolar spilanthol elicited little to no response in taste bud cells or trigeminal neurons. These same perithreshold concentrations of spilanthol significantly enhanced responses to NaCl (140 and 200 mM) in taste bud cells. Trigeminal neurons, however, exhibited response enhancement only at the highest concentrations of NaCl and spilanthol tested. Using a combination of potassium depolarization, immunohistochemistry, and Trpm5-GFP and Tas1r3-GFP mice to characterize taste bud cells by type, we found spilanthol enhancement of NaCl responses most prevalent in NaCl-responsive type III cells, and commonly observed in NaCl-responsive type II cells. Our results indicate that spilanthol enhances NaCl responses in taste bud cells and point to a family of compounds that may have utility as salty taste enhancers.

Introduction

Table salt (NaCl) is an important component of flavor and an essential nutrient critical for cellular homeostasis in humans and mammals. However, long-term overconsumption of NaCl has been linked to increased hypertension and cardiovascular disease-related morbidity (Taylor et al., 2011; Yang et al., 2011; Whelton et al., 2012). In the United States, about 78 million adults have hypertension, and more than 80 million people have different forms of cardiovascular disease (Roger et al., 2011). Experiments and clinical observation reveal that reducing dietary NaCl intake has major beneficial effects on blood pressure and cardiovascular health (Ha, 2014). A promising adjunct to the strategy of reducing dietary NaCl intake are efforts to identify food additives that can increase taste sensitivity to NaCl, thereby reducing the amount of NaCl necessary to achieve desirable levels of perceived saltiness.

Spilanthol is an unsaturated fatty acid amide produced by a number of plants, including several plants of the genus Acmella, most notably Acmella oleracea a spice more commonly known as jambu or paracress (Nakatani and Nagashima, 1992; Ramsewak et al., 1999; Spelman et al., 2011; Barbosa et al., 2016; Franca et al., 2016). Extracts of the jambu plant are used as a food-flavoring agent in the cuisines of many countries worldwide (Efsa Panel on Food Contact Materials and Processing, 2011; World Health, 2011). Spilanthol and several structurally related unsaturated fatty acid amides induce a variety of somatosensory sensations, including pungency, tingling, buzzing, numbing, mouthwatering, and/or cooling sensations (spilanthol [Nakatani and Nagashima, 1992; Gyekis et al., 2012; Barbosa et al., 2016]; sanshool [Bryant and Mezine, 1999; Sugai et al., 2005a, 2005b]; isobutylalkylamide [IBA] [Albin and Simons, 2010; Tulleuda et al., 2011]). For one of these amides, sanshool, these sensations have been attributed to activation of mechanosensitive trigeminal neurons through inhibition of 2-pore-domain potassium (K2P) channels (Bautista et al., 2008; Lennertz et al., 2010; Tsunozaki et al., 2013). On the basis of similarities in chemical structure and psychophysical effect, we hypothesized that spilanthol may also inhibit K2P channels and lead to enhanced gustatory responses in taste receptor cells.

Blocking K⁺ leak currents through K2P channels increases membrane resistance and induces depolarization in most cells. In some neurons, this depolarization is sufficient to induce action potential firing. Even when K2P channel inhibition is insufficient to directly activate cells, the subthreshold depolarization and increased membrane resistance combine to make cells more sensitive to subsequent depolarizing stimuli. For example, IBA, a sanshool derivative that blocks TRESK family K2P channels, was shown to sensitize responses in dorsal root ganglion neurons (Tulleuda et al., 2011). And in the taste system, inhibition of the K2P leak channels TREK1 (KCNK2) and TREK2 (KCNK10) in sour taste cells enhanced responses to acidic stimuli (Richter et al., 2004). Whether spilanthol could similarly act on K2P leak currents present in salt-sensitive taste cells (Lin et al., 2004; Richter et al., 2004) and thus regulate sensitivity to sodium salts in taste bud cells (TBCs) and trigeminal sensory neurons is an open question.

Using calcium imaging of mouse sensory cells, we examined whether spilanthol sensitized TBC and trigeminal sensory neuron responses to NaCl. Sub- to perithreshold concentrations of spilanthol significantly enhanced the sensitivity and response magnitude to NaCl stimuli in the majority of NaCl-responsive type III TBCs and in more than half of NaCl-responsive type II TBCs. Trigeminal neurons were notably less sensitive to spilanthol, exhibiting significant response enhancement only at the highest concentrations of NaCl and spilanthol tested. These results suggest that low concentrations of spilanthol may be capable of selectively enhancing taste-related NaCl responses without inducing the less desirable numbing and tingling sensations carried by the trigeminal pathway.

Experimental methods and materials

Materials

Tyrode’s solution contains (in mM): 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), and 1 Na-pyruvate. Ca²⁺-free Tyrode’s solution contains (in mM): 140 NaCl, 5 KCl, 10 glucose, 10 HEPES, 1 Na-pyruvate, and 2 egtazic acid (EGTA). An equimolar substitution of NMDG.Cl for NaCl was used to make a low-NaCl Tyrode’s variant (in mM): 30 NaCl, 110 N-methyl-D-glucamine chloride (NMDG.Cl), 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, and 1 Na-pyruvate. Ca²⁺-free low-NaCl Tyrode’s solution contains (in mM): 30 NaCl, 110 mM NMDG, 5 KCl, 10 glucose, 10 HEPES, 1 Na-pyruvate, and 2 EGTA. All solutions were adjusted to pH 7.4 and the osmolarity was 320 mOsmol. For NaCl salt stimuli, we used either Tyrode’s solution (140 mM NaCl) or Tyrode’s solution with additional NaCl added to achieve the indicated concentration (200 and 250 mM NaCl). The 40-mM KCl stimulus was created by making an equimolar substitution of 35 mM KCl for NMDG.Cl in the low-NaCl Tyrode’s variant. Reagents used with trigeminal neuron dissociation included the following: Hank's balanced salt solution (HBSS), Ca²⁺- and Mg²⁺-free HBSS, and collagenase A (Roche); Dulbecco's modified Eagle medium (DMEM), penicillin/streptomycin (PS), and trypsin (Life Technologies); poly-d-lysine and laminin. Unless otherwise specified, all compounds were from Sigma-Aldrich, Inc. Other reagents used in this study include phosphate-buffered saline (PBS) (Thermo Scientific), Superblock buffer (Thermo Scientific), 40 mM KCl (used as a depolarizing stimulus for testing neuron viability and identifying taste cell subtypes), 3 and 6 µM spilanthol (>99% purity by high performance liquid chromatography (HPLC); Ogawa & Co. Ltd.), 1 mg/mL concanavalin A (ConA; Vector Laboratories) in 50 mM N-(2-Acetamido)-2-aminoethanesulfonic acid (ACEs) pH 8, and Fura2 acetoxymethylester (Fura2-AM) and pluronic F127 (both Invitrogen).

Animals and taste tissues

C57BL/6 mice were obtained from Charles River Co. (IMSR Cat# CRL:27, RRID:IMSR_CRL:27). Tas1r3-GFP and Trpm5-GFP mice were provided by Dr. Robert Margolskee, Monell Chemical Senses Center. The use and handling of animals were performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the National Institutes of Health guide (NIH Publications 80-23, revised 1978). The experimental protocols were approved by the institutional animal care and use committee of Monell Chemical Senses Center, and all efforts were made to reduce the number of animals used and minimize animal suffering.

Preparation of dissociated TBCs

For the acute preparation of mouse TBCs, 2- to 3-month-old mice were killed by CO₂ asphyxiation followed by cervical dislocation. The tongues were immediately excised and transferred into Tyrode’s solution. An enzyme mixture containing 1 mg/mL elastase (Worthington Biochemical Corporation), 2.5 mg/mL Dispase II (Roche), and 1 mg/mL trypsin inhibitor (Worthington Biochemical Corporation) dissolved in Tyrode’s solution was injected under the lingual epithelium using a 30-gauge needle. Following incubation at room temperature for 12–16 min the lingual epithelium was peeled and washed with Tyrode’s solution. The peeled epithelium was then incubated in Ca²⁺-free Tyrode’s solution at room temperature for 45 min. Individual taste cells and clumps of cells from the circumvallate papilla were collected from the epithelium using gentle suction with a fire-polished glass capillary (pore diameter ~40 µm) and plated immediately onto coverslips (22 × 40 mm, No. 0; Thomas Scientific) or gridded etched coverslips coated with ConA.

Preparation of trigeminal neurons

Mouse trigeminal ganglia were removed and initially placed into Ca²⁺- and Mg²⁺-free HBSS–PS solution until all tissues were collected. The ganglia were minced and incubated at 37 °C for 15 min in 5 mL of 0.05% trypsin in Ca²⁺- and Mg²⁺-free HBSS–PS solution. To stop the trypsin digestion, 10 mL HBSS–PS solution was added and the sample was centrifuged at 300 × g for 3 min. The pellet was resuspended in 5 mL of HBSS–PS containing 1 mg/mL collagenase A and incubated at room temperature for 20 min. The tissue was then gently triturated, centrifuged again, and the pellet gently resuspended in 0.5 mL DMEM. Neurons were harvested from the supernatant after 30 s of settling and were plated onto laminin/poly-d-lysine-coated glass coverslips or similarly treated 96-well plates. Neurons were incubated at 37 °C in 5% CO₂ for 1 h or overnight before imaging.

Experimental design and statistical analysis

Cellular responses were measured using ratiometric calcium imaging techniques as previously described (Inoue and Bryant, 2005). Acutely isolated mouse TBCs or trigeminal neurons were loaded with 5 µM Fura2-AM and 8 µL of 10% pluronic F127 in 1 mL of Tyrode’s solution for 1 h at room temperature. Coverslips with cells were set in a recording chamber and constantly superfused with low-NaCl Tyrode’s solution. The reduced concentration of sodium in the low-NaCl Tyrode’s perfusion solution (30 mM vs. 140 mM in standard Tyrode’s solution) was chosen to enable measurement of responses to 140 mM NaCl. Pilot experiments determined that complete elimination of sodium rendered taste cells unstable or nonviable before complete experiments could be performed.

Superfusion was controlled by a valve controller (VC-8; Warner) and peristaltic pump (Perimax 12; SPETEC). Stimulation duration was 30 s, and rinsing time was 3 min at 3.2 mL/min perfusion rate. Pairs of images (excitation: 340 and 380 nm; emission: 510 nm) were acquired every 5 s. The calcium imaging system consisted of a Lambda 10-2 optical control system (Sutter Instrument Co.), an Olympus IX70 microscope, and a MicroMax RS camera (Roper Scientific Inc.). The average fluorescence ratio, F₃₄₀/F₃₈₀, an index of [Ca²⁺]_i, was calculated for each cell (regions of interest drawn manually) using MetaFluor software (Molecular Devices; RRID:SCR_014294). All TBCs and trigeminal neurons that took up the Fura2-AM dye were included in initial digital analyses. Cells with baseline [Ca²⁺]_i that was highly variable or that drifted significantly over time were identified by visual inspection and excluded from further analyses.

Experimental stimulation consisted of a first stimulus of NaCl perfused over the cells followed by spilanthol (3 or 6 µM) presented alone and then by a mixture of the initial concentration of NaCl plus spilanthol. Response magnitudes were measured as the difference between the peak magnitude during the response window (90 s following presentation of stimulus) minus the mean baseline fluorescence ratio (calculated from the 50 s preceding initiation of stimulation). A positive response to a stimulus was defined as a response magnitude >0.01 fluorescence ratio units (arbitrary units, F_{340/380 nm}). We empirically determined this threshold by identifying 10 cells that responded robustly to multiple stimuli but whose baselines fluctuated noticeably more than the majority of cells in our data set. We quantified these fluctuations by calculating the standard deviation (SD) of a contiguous 120 s of baseline values for each cell. We determined that 0.01 ΔF_{340/380 nm} was >10× the SD of baseline fluorescence calculated from these more variable cells, whereas the magnitude of stimulus induced calcium responses in our recordings was well above 0.01 ΔF_{340/380 nm}. Thus, we chose 0.01 ΔF_{340/380 nm} as a general threshold for distinguishing stimulus responses from baseline fluctuations.

The effect of spilanthol on response magnitude was quantified as the difference in response magnitude elicited by stimulation with NaCl + spilanthol versus NaCl alone. We used an empirically determined threshold of >0.03 ΔF_{340/380 nm} to define spilanthol-mediated response enhancement. This threshold is more stringent than the >0.01 F_{340/380 nm} threshold we used to determine stimulus responses because response magnitudes to repeated presentations of a stimulus are more variable than fluctuations in baseline fluorescence. The limited number of stimuli that can be presented in calcium imaging experiments using our acutely isolated cell preparations made statistical determination of a threshold for spilanthol-mediated response enhancement impractical.

To compare the magnitude of NaCl responses across TBCs and trigeminal neurons (Figure 1), the response magnitudes to NaCl stimuli recorded for a given cell were normalized to that cell’s average response magnitude to 40 mM KCl (ΔF_Norm). Only TBCs and trigeminal neurons that responded (ΔF_{340/380 nm} > 0.01 AU) to 40 mM KCl and at least one of the NaCl concentrations tested were included in the data for Figure 1.

Figure 1.

TBCs and trigeminal neurons responded in a concentration-dependent manner to NaCl. Data represent mean calcium response magnitudes (±standard error of mean) of trigeminal neurons and TBCs to 140 mM NaCl (n = 26 trigeminal; n = 52 TBCs), 200 mM NaCl (n = 26 trigeminal; n = 54 TBCs), and 250 mM NaCl (n = 19 trigeminal; n = 54 TBCs). Only cells that exhibited responses >0.01 ΔF_340/380 to both 40 mM KCl and at least one of the NaCl concentrations tested were included in these analyses. NaCl response magnitudes in a given cell were normalized to its average KCl response. NaCl-evoked calcium responses were significantly larger in TBCs compared with trigeminal neurons at all concentrations tested (140 mM NaCl: t(57) = 5.21, P = 2.70E–06; 200 mM NaCl: t(76) = 4.07, P = 0.00011; 250 mM NaCl: t(71) = 4.93, P = 5.27E–06, 2-tailed, two sample unequal variance).

If a stimulus was presented more than once to a given cell, the response magnitudes for each presentation were averaged, and this average value was used for quantification. Statistical tests were performed using this quantified data.

Immunohistochemistry

Standard immunohistochemical techniques were used. Briefly, isolated TBCs on coverslips were washed 2× with PBS (pH 7.3) and fixed in freshly prepared 2% paraformaldehyde in PBS at room temperature for 10 min. After washing in PBS, nonspecific binding was blocked with SuperBlock buffer in PBS at room temperature for 1–2 h. A rat monoclonal anti-neural cell adhesion molecule (NCAM; dilution 1:250; Chemicon; No. MAB310) was used to identify type III taste cells. This rat IgG_2a antibody (neural BSP₂ identical to NCAM) was raised against a glycoprotein fraction from neonatal mouse brain and recognized the neural cell surface triplet of glyocoproteins via immunoprecipitate results, with apparent molecular weights of 180, 140, and 120 kDa (Hirn et al., 1981). This antibody recognized mouse type III taste cells during development and after denervation (Uchida et al., 2003). The antibody was diluted in blocking solution and cells were incubated overnight at 4 °C. On the following day, after three 5-min rinses in PBS containing 0.3% Triton X-100, cells were incubated in Alexa Fluor 594 donkey anti-rat antibodies (Thermo Fisher Scientific Cat# A-21209, RRID:AB_2535795) (1:500) at room temperature for 1 h and then washed 3× with PBS. The immunofluorescence images were captured with an Olympus fluorescence microscope (40×, Olympus IX70). Negative controls were imaged using the same acquisition settings (i.e., exposure time and gain) as the corresponding experimental samples. No adjustments of brightness or contrast were made to the positive and negative control images.

Results

The primary goal of this study was to determine if sensory cell responses to NaCl were enhanced by spilanthol. Spilanthol is one of several structurally similar unsaturated alkylamides, including sanshool and IBA (Figure 2), that induce tingling, buzzing, and other somatosensory sensations. Sanshool, the most studied of these unsaturated alkylamides, activates somatosensory mechanoreceptor neurons by inhibiting the potassium leak currents mediated by K2P channels, specifically KCNKs 3, 9, and 18 (Bautista et al., 2008; Lennertz et al., 2010). We hypothesized that spilanthol, like sanshool, could depolarize cell membranes, likely through inhibition of K2P family channels, and this depolarization would cause an enhancement of responses to subsequent depolarizing stimuli, specifically NaCl, in trigeminal neurons and TBCs. We examined responses in TBCs and trigeminal neurons because both have been implicated in encoding the orosensory attributes of NaCl, and thus both may contribute to the potential effects of spilanthol on NaCl perception. Fura2 calcium imaging was used to measure responses to NaCl and spilanthol in mouse taste cells (isolated from the circumvallate papillae) and trigeminal neurons (isolated from the trigeminal ganglia). To limit any potential desensitization of NaCl response pathways, a low-NaCl variant of Tyrode’s solution (30 mM NaCl + 110 mM NMDG.Cl) was used as bath solution.

Figure 2.

Chemical structures of 3 unsaturated alkylamides that produce similar somatosensory sensations in the mouth. Sanshool and IBA are known to inhibit members of the K2P channel family expressed by subsets of trigeminal neurons and other sensory cells. Spilanthol is hypothesized to also interact with K2P channels based on similarities to sanshool and IBA in both chemical structure and induced somatosensory sensations.

Responses to NaCl and spilanthol in orosensory cells

Both isolated TBCs and trigeminal neurons responded in a concentration-dependent manner to NaCl (Figure 1). Taste cells were more sensitive to NaCl than trigeminal neurons, which is in agreement with findings from perceptual and nerve recording experiments (Sostman and Simon, 1991). Tested alone, micromolar concentrations of spilanthol (3 and 6 µM) evoked little to no calcium response in either trigeminal neurons (Figure 3) or TBCs (Figure 4). The vehicle for spilanthol, 0.1% EtOH, also elicited no calcium response in either trigeminal neurons (ΔF_{340/380 nm} = –0.010, n = 19) or taste cells (ΔF_{340/380 nm} = –0.0047, n = 9). For spilanthol to induce a response, its concentration would need to be high enough to cause K2P channel inhibition sufficient to depolarize a cell past the threshold necessary to activate a signal transduction cascade. For trigeminal neurons and most/all NaCl-responsive taste cells, the relevant signal transduction cascades are likely triggered by activation of voltage-gated sodium channels (Medler et al., 2003; Bigiani, 2016). Although the low concentrations of spilanthol we tested were not strong enough to induce a response on their own, we predicted that the subthreshold depolarization and the increase in membrane resistance caused by low-level inhibition of K2P channels by spilanthol would enhance responses to subsequent depolarizing stimuli.

Figure 3.

Trigeminal neuron NaCl responses are not affected by spilanthol. (A, B) Exemplar calcium imaging data from 2 NaCl-responsive trigeminal neurons demonstrating the characteristic lack of responsiveness to spilanthol (Spil., 3 and 6 µM) and the absence of spilanthol-mediated NaCl response enhancement. (C) Summary data of trigeminal neuron calcium responses to 140 mM NaCl (C) and 200 mM NaCl (D). Also plotted are responses to 3 and 6 µM spilanthol +/– NaCl. Spilanthol significantly enhanced NaCl responses only at the highest concentrations tested: 200 mM NaCl + 6 µM spilanthol (140 mM NaCl + 3 µM spilanthol: t(14) = 1.08, P = 0.15; +6 µM spilanthol: t(20) = –0.16, P = 0.44; 200 mM NaCl + 3 µM spilanthol: t(11) = –1.24, P = 0.12; + 6 µM spilanthol: t(26) = –2.63, P = 0.0071).

Figure 4.

Spilanthol enhances NaCl response magnitude and sensitivity in TBCs. (A, B) Exemplar calcium imaging data from 2 isolated taste cells that responded to NaCl (140 and 200 mM) and exhibited spilanthol (Spil., 3 and 6 µM) mediated enhancement of NaCl responses. (C) Responses to 140 mM NaCl were significantly enhanced by both 3 µM (t(14) = –2.84, P = 0.0066, paired 1-tailed t-test) and 6 µM (t(86) = –5.24, P = 5.53E–07, paired 1-tailed t-test) concentrations of spilanthol. (D) Responses to 200 mM NaCl were enhanced by 6 µM (t(85) = –4.62, P = 6.80E–06, paired 1-tailed t-test) but not 3 µM (t(29) = –1.18, P = 0.12, paired 1-tailed t-test) spilanthol. Response magnitudes in panels C and D represent the peak response minus the baseline average fluorescence ratio (F_{340/380 nm}). (E) Exemplar data from a TBC that did not respond to 140 mM NaCl presented alone but did respond to 140 mM NaCl when spilanthol was added.

Effect of spilanthol on NaCl sensitivity in TBCs and trigeminal neurons

To assess spilanthol’s potential as a salty taste enhancer, we tested whether sub- to perithreshold concentrations of spilanthol (3 and 6 µM) could enhance responses to NaCl. In NaCl-responsive trigeminal neurons, 3 µM spilanthol had no significant effect on the magnitude of responses to 140 or 200 mM NaCl, whereas 6 µM spilanthol significantly enhanced the magnitude of responses to 200 mM NaCl, but not 140 mM NaCl (Figure 3). Furthermore, no trigeminal neurons that were initially unresponsive to 140 or 200 mM NaCl became responsive in the presence of spilanthol. Taken together these data indicate that trigeminal neuron NaCl responses are relatively insensitive to micromolar concentrations of spilanthol. The predicted enhancement of NaCl response magnitudes only emerged when perithreshold concentrations of spilanthol were combined with high concentrations of NaCl.

Unlike trigeminal neurons, the majority of NaCl-responsive TBCs exhibited significantly greater magnitude calcium responses to NaCl in the presence of spilanthol (Figure 4). Among all TBCs tested, 15.1% (n = 184/1222) responded to 140 and/or 200 mM NaCl. Of the 184 NaCl-responsive TBCs, 72.8% (n = 134/184) had greater magnitude responses to the mixture of NaCl + spilanthol compared with NaCl alone. An additional subset of TBCs (n = 58) that did not respond to the initial 140 or 200 mM NaCl stimulus became responsive to NaCl in the presence of spilanthol (Figures 4E and 7A). These data indicate that spilanthol can both increase the sensitivity of TBCs to NaCl and enhance the magnitude of NaCl-evoked responses.

Figure 7.

Extracellular calcium influx is necessary for NaCl responses and spilanthol enhancement in isolated taste cells. (A) Exemplar calcium imaging data from a type III cell demonstrating the necessity of extracellular calcium for spilanthol-mediated NaCl response enhancement. This cell had little to no response to 200 mM NaCl but responded strongly to 200 mM NaCl + 6 µM spilanthol. NaCl + spilanthol responses are eliminated when the cell is bathed in calcium-free Tyrode’s solution, but recover once standard (low-NaCl Tyrode’s solution) bath solution is reintroduced. (B). The average magnitude of calcium responses to 200 mM NaCl and 200 mM NaCl + 6 µM spilanthol recorded from 32 NaCl-responsive taste cells tested before, during and following washout of calcium-free Tyrode’s bath solution. No responses to the NaCl stimuli were observed in the absence of extracellular calcium.

Salt responses in taste cell subtypes

Our data indicated that low concentrations of spilanthol (3 and 6 µM) might be used to selectively enhance NaCl responses in TBCs without significantly impacting the responsivity of trigeminal neurons. To get an idea of how spilanthol at these concentrations might impact the perceived taste of NaCl, we tested for salt responses and spilanthol enhancement in identified subpopulations of taste cells. Taste cells are generally grouped into types (I, II, and III) based on morphology and characteristic patterns of gene expression (Bachmanov and Beauchamp, 2007; Chaudhari and Roper, 2010; Liman et al., 2014). Within the type II and type III groups of taste cells are subpopulations specialized for the detection of 1, or sometimes 2, taste qualities (Zhang et al., 2003; Tomchik et al., 2007; Yoshida, Miyauchi et al., 2009; Oka et al., 2013; Lewandowski et al., 2016; Yoshida and Ninomiya, 2016). To determine which subpopulations of taste cells exhibited spilanthol-induced enhancement of NaCl responses, we recorded responses to KCl depolarization and to NaCl +/– spilanthol in subpopulations of taste cells identified using physiological and/or immunohistochemical techniques.

Classification of taste cell types

Type II cells were identified by GFP fluorescence in experiments conducted using Tas1r3-GFP and Trpm5-GFP transgenic mice. TAS1R3 is expressed by sweet and/or umami-responsive type II cells (Zhao et al., 2003); TRPM5 is expressed by sweet, umami, and bitter-responsive type II cells (Perez et al., 2002; Zhang et al., 2003; Bachmanov and Beauchamp, 2007). Type III cells were identified using a combination of molecular and physiological criteria. In a small number of experiments using Tas1r3-GFP and Trpm5-GFP mice, we conducted post hoc immunohistochemistry for the type III cell marker NCAM (Nelson and Finger, 1993; DeFazio et al., 2006; Sukumaran et al., 2017). In these experiments, cells were classified as type III if they were immunopositive for NCAM and did not express GFP. Because molecular and physiological studies have shown that most KCl-responsive taste cells are type III cells (DeFazio et al., 2006; Huang et al., 2007; Huang et al., 2009; Lewandowski et al., 2016), responsivity to KCl is regularly used to help physiologically identify type III cells. However, previous research (Oka et al., 2013; Lewandowski et al., 2016) and our own data set have detected KCl responses in a small subpopulation of type II taste cells. In experiments using Tas1r3-GFP and Trpm5-GFP mice, GFP fluorescence could be used to identify and exclude most type II cells, so GFP-negative cells that responded to KCl depolarization were classified as type III cells with reasonable confidence. In addition, we chose to putatively classify cells from C57BL/6 wild-type mice as type III if they responded to KCl depolarization. It is highly likely that the group of putative type III cells (n = 147) identified in experiments using C57BL/6 mice includes some KCl-responsive type II cells. However, any misclassified cells would represent only a small fraction of the total population of type III TBCs analyzed in this study (n = 317).

Any cells that did not meet the aforementioned criteria for classification as type II or type III TBCs were categorized as “ambiguous” and analyzed separately. These include 11 cells that were both Tas1r3-GFP and NCAM-positive, 8 cells that were GFP- and NCAM-negative, and all GFP-negative cells that did not respond to KCl depolarization. These “ambiguous” cells will be largely excluded from the following discussion as the vast majority, 96.2% (n = 580/603), did not respond to any of the stimuli tested.

Distribution of NaCl and KCl responses among taste cell types

We presented 1222 isolated taste cells with NaCl and KCl and measured their responses using calcium imaging. Although the primary goal of these experiments was to assess the effect of spilanthol on NaCl responses in taste cells, this data set represents the only large scale survey to date of NaCl and KCl responses in isolated taste cells. Studies of breadth of tuning in intact taste buds find that, among the five basic taste qualities, responses to NaCl are the least common, and cells responsive to NaCl are highly likely to also respond to one or more other taste qualities (Caicedo et al., 2002; Tomchik et al., 2007; Yoshida, Miyauchi et al., 2009). In the intact taste bud, paracrine signaling between taste cells can result in taste cells responding to tastants for which they do not express receptors; for example, type III cells commonly respond to bitter tastants despite lacking bitter receptors (Chandrashekar et al., 2000; Tomchik et al., 2007). Using isolated taste cells eliminates this paracrine signaling and ensures that only those taste cells that express the necessary channels/receptors to detect a stimulus will respond when that stimulus is presented. To gain a better understanding of the native response properties of different taste cell types to salts, we analyzed the distribution of NaCl and KCl responses among the subpopulations of isolated taste cells in our data set (Figure 5A–C).

Figure 5.

Summary of responses to NaCl and KCl depolarization and spilanthol enhancement in identified taste cell types. Pie chart summaries showing the proportion of cells classified as type II (A), type III (B), and ambiguous (C) (see text for details on cell type classification) that responded to NaCl (140 or 200 mM), KCl depolarization (40 mM), both NaCl and KCl, or neither stimulus. Note that a response to KCl depolarization was one of the criteria used to classify most cells as type III. The distribution of KCl responses in type III cells identified based on nonphysiological criteria can be seen in Figure 6C. Also note that cells that were not GFP- or NCAM-positive and did not respond to NaCl or KCl were classified as ambiguous. (D) Summary graph of spilanthol enhancement broken down by cell type and physiological profile. The percentage of type II, type III, and ambiguous cells exhibiting spilanthol enhancement of NaCl responses is plotted for all recorded cells in each category (left), only those cells that responded to NaCl (middle), and for cells that did not respond to the initial NaCl stimulus (right). This last category is the percentage of cells that were unresponsive to NaCl (140 or 200 mM) tested alone but became responsive to the NaCl stimulus in the presence of spilanthol. Numbers above the bars indicate the total number of cells within each category. Type III cells were both the most likely to exhibit spilanthol enhancement of existing NaCl responses and the most likely to become responsive to previously subthreshold NaCl concentrations in the presence of spilanthol. (E) Bar graph of the number of cells that responded to NaCl only in the presence of spilanthol broken into 2 groups based on whether the cell also responded to KCl depolarization. In keeping with the hypothesis that spilanthol inhibits potassium channels, nearly all cells in which spilanthol was observed to reduce NaCl-response thresholds also responded to KCl depolarization. Bars are subdivided to indicate the number of plotted cells belonging to different subpopulations of classified taste cells: ambiguous (light gray), type II Tas1r3-GFP+ (light blue), type II Trpm5-GFP+ (dark blue), type III NCAM+ (bright red), type III KCl-responsive and Trpm5-GFP- (orange), all other type III (dark red).

NaCl and KCl responses in type II cells

Among the GFP-positive type II TBCs in our data set, 15.5% (n = 47/302) responded to NaCl and 24.8% (n = 75/302) responded to KCl (Figure 5A). The incidence of NaCl responses was similar for Tas1r3-GFP cells (15.0%, n = 26/173) and Trpm5-GFP cells (16.3%, n = 21/129). The overall percentage of NaCl-responsive type II cells that we observed is higher than has been reported from studies of intact taste buds. However, the proportion of Tas1r3-GFP and Trpm5-GFP cells responsive to NaCl fits with the results of prior studies, which find that, among type II cells, NaCl responses are most commonly observed in sweet-responsive cells, which express both Tas1r3 and Trpm5 (Zhang et al., 2003; Liman et al., 2014).

Responses to KCl were slightly more prevalent among Tas1r3-GFP cells (28.9%, 50 of 173) than Trpm5-GFP cells (19.4%, 25 of 129). The few studies that have examined KCl responses in type II cells report that they are primarily found in the subpopulation of bitter-responsive type II cells that also respond to amiloride-insensitive (AI) salt stimuli (Oka et al., 2013; Lewandowski et al., 2016). Bitter-responsive type II cells do not express TAS1R3, so the prevalence of KCl responses in Tas1r3-GFP cells was unexpected.

Type II cells that responded to NaCl frequently responded to KCl as well. This was particularly true for NaCl-responsive Tas1r3-GFP cells where 88.5% (n = 23/26) also responded to KCl, compared with 52.4% (n = 11/21) for Trpm5-GFP cells. More type II cells responded only to KCl than responded only to NaCl. Among KCl-responsive type II cells, 46.0% (n = 23/50) of Tas1r3-GFP and 44.0% (n = 11/25) of Trpm5-GFP cells also responded to NaCl. The use of circumvallate taste buds, which do not contain amiloride-sensitive (AS) salt taste cells in mice (Ninomiya, 1998), may have contributed to the relative rarity of cells responsive to NaCl but not KCl.

NaCl and KCl responses in type III cells

A meaningful analysis of KCl responses for the entire population of type III cells in our data set is not possible because a response to KCl depolarization was one of the criteria used to classify the majority of these cells as type III. However, a small number of type III cells (n = 29) were identified based exclusively on their molecular profile, specifically immunopositivity for NCAM combined with a lack of Tas1r3-GFP or Trpm5-GFP fluorescence (Figure 6). These cells enable us to test the validity of using KCl responses as a physiological marker of type III cells. Of the 29 cells classified as type III based on NCAM immunopositivity, 28 responded to KCl depolarization (Figure 6C). This agrees with findings from previous studies and supports our use of KCl responses as physiological criteria for identifying type III cells. The NCAM+ type III cells were also notable for being the most likely to respond to NaCl, with 75.9% (n = 22/29) responding to 140 mM and/or 200 mM NaCl. Furthermore, the remaining 7 NCAM+ type III cells that did not initially respond to NaCl became responsive to NaCl in the presence of spilanthol (Figure 6C). Figure 6A and B shows exemplar data from a NCAM+ type III taste cell that exhibited spilanthol-induced NaCl response enhancement.

Figure 6.

Spilanthol enhanced NaCl responses in most NCAM-positive (type III) TBCs. (A) Immunostaining of an NCAM-positive TBC isolated from the circumvallate papillae of a Tas1r3-GFP mouse. Panels show bright field, GFP fluorescence, NCAM fluorescence, and merged images of the NCAM-positive, GFP-negative TBC. (B) Calcium imaging of the cell shown in panel A. Like the majority of type III cells identified by NCAM immunoreactivity, this cell responded to 140 mM NaCl and exhibited spilanthol mediated NaCl response enhancement. (C) Pie chart showing the distribution of NaCl and KCl responses recorded from 29 NCAM+ type III cells. White hatched lines mark the populations of cells where spilanthol enhanced NaCl responses for the NaCl-only (light gray) and NaCl + KCl responsive (black) groups. For the KCl-only group (medium gray), which did not respond to NaCl presented alone, hatched lines indicate cells where addition of spilanthol enabled responses to the previously subthreshold NaCl stimulus. NCAM+ type III cells were more likely to respond to NaCl and exhibit spilanthol-mediated NaCl response enhancement than the other subpopulations of taste cells examined.

Among type III cells identified using criteria other than NCAM immunopositivity, 34.0% (n = 98/288) responded to NaCl. All together, 37.9% (n = 120/317) of type III cells were NaCl responsive (Figure 5B). Estimates of NaCl responsivity among type III cells in intact taste buds vary considerably (Tomchik et al., 2007; Yoshida, Horio et al., 2009; Yoshida, Miyauchi et al., 2009), but 37.9% would be slightly above the higher estimates.

Spilanthol enhancement of salt responses in taste cell subtypes

To better understand the effect of spilanthol on NaCl responses in taste cells, we compared calcium responses to NaCl and NaCl + spilanthol in taste cell subtypes (Figure 5D). Among type II taste cells, spilanthol enhanced NaCl responses in 51.1% (n = 24/47) of NaCl-responsive taste cells. In addition, 9 cells that did not respond to NaCl presented alone became responsive to NaCl in the presence of spilanthol. Spilanthol enhancement of NaCl responses was slightly more prevalent among Tas1r3-GFP cells (57.7%, n = 15/26) than Trpm5-GFP cells (42.9%, n = 9/21). However, of the 9 type II cells that only responded to NaCl in the presence of spilanthol, 8 were Trpm5-GFP cells (Figure 5E).

Spilanthol had a notably stronger effect on both the magnitude and threshold of NaCl responses in type III cells than in type II cells. Among NaCl-responsive type III taste cells, 81.7% (n = 98/120) exhibited spilanthol enhancement, and 39 initially unresponsive type III cells became responsive to NaCl in the presence of spilanthol.

To test whether spilanthol-mediated reductions in NaCl response thresholds were occurring preferentially in particular populations of TBCs, we focused our analysis on the TBCs that only responded to NaCl in the presence of spilanthol (Figure 5E). The majority of these cells were type III TBCs (61.7%, n = 39/58), with the rest distributed relatively equally between type II TBCs (15.5%, n = 9/58) and ambiguous cells (17.2%, n = 10/58). These results are similar to our population-level analyses that found that both NaCl responsivity and spilanthol enhancement were most common in type III cells. Of the 9 type II cells where spilanthol reduced NaCl response thresholds, 8 were Trpm5-GFP cells; the significance of this, if any, is unclear because the population of Trpm5-GFP cells will include many Tas1r3-expressing cells.

In support of our hypothesis that spilanthol is acting on K2P channels, 91.4% (n = 53/58) of the cells that responded to NaCl only in the presence of spilanthol also responded to KCl depolarization (Figure 5E). KCl-induced depolarization results primarily from the reversal of K+ currents through K+ leak channels.

Taken together these results show that spilanthol enhancement is common in all NaCl-responsive subpopulations of taste cells. However, type III cells are more likely than type II cells to 1) respond to NaCl, 2) exhibit spilanthol-mediated NaCl response enhancement, and 3) demonstrate an increased sensitivity to NaCl in the presence of spilanthol (seen in the greater percentage of type III cells with spilanthol-mediated reductions in NaCl response thresholds).

Role of external calcium in responses to NaCl and their enhancement by spilanthol

NaCl- and KCl-induced calcium responses in type III cells require the influx of extracellular calcium through voltage-gated calcium channels (Richter et al., 2003; Roberts et al., 2009). Multiple studies have found that type II cells lack voltage-gated calcium channels (Clapp et al., 2006; DeFazio et al., 2006; Roberts et al., 2009). In type II cells, signaling through tastant-responsive G protein-coupled receptors (GPCRs) results in release of calcium from intracellular stores (Chaudhari and Roper, 2010; Liman et al., 2014). It is currently unclear which pathways mediate KCl- and/or NaCl-evoked calcium responses in type II cells, and there are seemingly conflicting reports regarding the necessity of intracellular versus extracellular calcium. In isolated taste cells, removal of extracellular calcium eliminated responses to KCl depolarization in bitter-sensitive cells (Hacker et al., 2008); however, the bitter-sensitive cells in which this response pattern was observed lacked canonical type II cell markers. In another study using intact taste buds, disruption of the G protein signaling pathway, which is linked to intracellular calcium release, significantly reduced responses to KCl and NaCl in type II cells (Oka et al., 2013). Given this uncertainty, we set out to test the necessity of extracellular calcium for NaCl responses and spilanthol enhancement in type II and type III cells.

Removing extracellular calcium abolished responses to 200 mM NaCl in nearly all TBCs tested (n = 31/32; Figure 7). Moreover, in the absence of extracellular calcium, no changes in intracellular calcium were observed to the mixture of 6 µM spilanthol and 200 mM NaCl. The fact that enhancement does not occur in the absence of extracellular calcium suggests that whatever effect spilanthol has at or downstream of the initial transduction of NaCl, it is not directly linked to intracellular calcium release mechanisms.

Discussion

A number of complementary approaches, including regulation, education, and research, have been explored to mitigate the health risks associated with excessive salt intake in humans. An important component of these efforts is research to identify a non-sodium compound that can enhance responses to NaCl and/or directly activate salt-responsive taste cells. Development of a salt substitute or salty taste enhancer would have the significant advantage of reducing human NaCl consumption while maintaining desirable levels of perceived saltiness. One family of unsaturated alkylamides, which includes spilanthol and sanshool, has the potential to serve as salty taste enhancers. In this study, we examined the ability of spilanthol to modulate responses to NaCl in the TBCs and trigeminal neurons that mediate the orosensory attributes of salty taste. Both TBCs and trigeminal neurons exhibited robust NaCl-induced calcium responses, but only in TBCs were NaCl responses broadly enhanced by micromolar concentrations of spilanthol.

Trigeminal neuron responses to NaCl and spilanthol

The tingling and numbing sensations induced by higher concentrations of spilanthol and sanshool have been linked to activation of trigeminal nerve endings (Bautista et al., 2008). NaCl can also stimulate trigeminal nerve endings and act as an irritant in the mouth, though at higher concentrations than we tested (Sostman and Simon, 1991; Dessirier et al., 2001). However, in these previous studies, trigeminal nerve endings were likely protected from exposure to the full concentration of NaCl tested due to tight junctions that inhibit diffusion of Na⁺ and Cl^– ions through the lingual epithelium (Dando et al., 2015). Because isolated trigeminal neurons are not similarly protected, we anticipated that concentrations of NaCl appropriate for stimulating taste cells would also be effective stimuli in our trigeminal neuron preparation. Indeed, robust calcium responses were elicited in our trigeminal neurons by both 200 and 250 mM NaCl, and some trigeminal neurons even responded to 140 mM NaCl, the lowest concentration we tested.

Given that the K2P channel TRESK (KCNK18) is present on many trigeminal/dorsal root ganglion neurons (Dobler et al., 2007; Enyedi and Czirjak, 2015) and is inhibited by the sanshool derivative IBA (Tulleuda et al., 2011), we had predicted that spilanthol would enhance trigeminal neuron responses to NaCl. Micromolar concentrations of spilanthol tested alone were not effective in stimulating isolated trigeminal neurons. Nominal responses were occasionally observed to 6 µM spilanthol, suggesting that spilanthol was interacting, albeit weakly at these concentrations, with channels on trigeminal neurons. Despite this, however, significant spilanthol-mediated enhancement of NaCl-evoked calcium responses was only observed at the highest concentrations tested (200 mM NaCl + 6 µM spilanthol).

One possible explanation for NaCl-responsive trigeminal neurons being less sensitive to spilanthol enhancement than NaCl-responsive TBCs is that the irritation-related salt responses in the trigeminal nerve could be mediated by neurons that do not express alkylamide-sensitive K2P channels. Potentially relevant to this idea is evidence that the K2P channels most sensitive to sanshool, and thus likely spilanthol as well, are primarily found on mechanoreceptors and much less commonly on nociceptors (Bautista et al., 2008; Lennertz et al., 2010; Tsunozaki et al., 2013). Another possibility is that the level of K2P channel inhibition caused by the lower concentrations of spilanthol we tested was too weak to significantly impact the transduction pathway(s) mediating NaCl responses in trigeminal neurons.

Spilanthol enhancement of NaCl responses in TBCs

Over 70% of NaCl-responsive TBCs exhibited spilanthol-mediated enhancement of NaCl responses. Importantly, TBC responses to spilanthol itself were null or minimal indicating that NaCl-response enhancement was more than simply additive. Spilanthol also increased the sensitivity of some TBCs to NaCl, enabling them to respond to previously subthreshold concentrations of NaCl.

On the basis of similarity in psychophysical effect and chemical structure with sanshool, we hypothesized that spilanthol can inhibit K2P channels. Resting leak currents attributed to K2Ps are an important component of the outward currents determining the resting membrane potential in mammalian TBCs (Bigiani, 2001; Lin et al., 2004; Vandenbeuch et al., 2008). Expression of the following K2P channels has been detected in TBCs: KCNK1 (TWIK1), KCNK2 (TREK1), KCNK3 (TASK1), KCNK5 (TASK2), KCNK10 (TREK2), KCNK16 (TALK1) (Lin et al., 2004; Richter et al., 2004; Sukumaran et al., 2017). KCNK3 (TASK1) is reported to be the K2P channel most sensitive to sanshool (Bautista et al., 2008). KCNK9 (TASK3) and KCNK18 (TRESK) are also sanshool sensitive but have not been detected in taste tissue. Thus, spilanthol’s action on TBCs may be attributable to TASK1 (KCNK3) and/or other K2P channels for which sensitivity to unsaturated alkylamides has not yet been specifically determined.

At least 2 distinct transduction pathways mediate TBC responses to NaCl, and NaCl responses have been observed in multiple subpopulations of taste cells, some of which also encode information related to other taste qualities (e.g., sweet, bitter, and sour) (Chandrashekar et al., 2010; Oka et al., 2013; Roper, 2015; Lewandowski et al., 2016). Despite some recent progress, it remains unclear what role individual subpopulations of NaCl-responsive TBCs play in mediating the different sensory aspects of salty taste (Vandenbeuch et al., 2008; Chandrashekar et al., 2010; Oka et al., 2013; Roper, 2015; Lewandowski et al., 2016). Behavioral preference tests from mice lacking AS responses found a significant reduction in NaCl preferences (Chandrashekar et al., 2010); whereas similar data from mice lacking AI responses showed a loss of aversion to higher concentrations of NaCl (Oka et al., 2013). One interpretation of these results is that the AS and AI pathways encode the appetitive and aversive components of salty taste, respectively. However, the concentrations of NaCl that humans find most palatable (Mattes et al., 1983; Villela et al., 2014) are high enough to activate both the AS and AI pathways. We believe an equally valid interpretation of the mouse behavioral results is that the AS pathway is necessary for distinguishing Na+ from non-Na+ salts whereas the AI pathway provides information on the concentration of the Na+ salt stimulus. This interpretation fits with human perceptual data showing that larger anion sodium salts, which should elicit normal AS responses but markedly reduced AI responses (Elliott and Simon, 1990; Ye et al., 1991; Kitada, 1995; Breza and Contreras, 2012), are perceived as significantly less salty than NaCl (Murphy et al., 1981; Schiffman et al., 1990; van der Klaauw and Smith, 1995). Given our current understanding of salty taste encoding, it is unclear which subpopulation(s) of NaCl-responsive TBCs would be the best target(s) for a salty taste enhancer. This is further complicated by the considerable uncertainty regarding how much of a role AS responses contribute to human salty taste perception (Desor and Finn, 1989; Breslin and Beauchamp, 1995; Ossebaard and Smith, 1995). Given what we do know, however, it is reasonable to hypothesize that, at least for humans, a compound that broadly enhances responses across all AI salt-responsive TBC populations could be an effective salty taste enhancer.

To determine the specificity of spilanthol’s effect on TBC NaCl responses, we examined responses to NaCl and/or spilanthol in different subpopulations of NaCl-responsive TBCs. Spilanthol enhancement was strongest and most prevalent in type III TBCs. Type III cells fire action potentials using the same collection of voltage-gated channels and synaptic vesicle machinery found in neurons (Roper and Chaudhari, 2017; Sukumaran et al., 2017). Thus, spilanthol enhancement is likely mediated by similar mechanisms to those identified in neurons, namely subthreshold depolarization caused by inhibition K+ leak currents through K2P channels (Bautista et al., 2008; Albin and Simons, 2010; Tulleuda et al., 2011). Further supporting this hypothesis, inhibition of K2P channels on type III cells has been previously shown to enhance their responses to acid (sour) stimuli (Richter et al., 2004). This mechanism of action for spilanthol could account for both the enhanced the magnitude of NaCl responses and the increased the sensitivity of type III cells to NaCl.

A notable number of type II TBCs exhibited responses to NaCl and spilanthol enhancement. Few studies have specifically examined NaCl responses in type II cells, but those that have report NaCl responses in a subset of sweet-responsive type II cells and a subset of bitter-responsive type II cells (Tomchik et al., 2007; Yoshida, Miyauchi et al., 2009; Oka et al., 2013). The percentage of NaCl and KCl responsive type II cells that we observed is higher than has been observed in intact taste buds where observations of salt responses in type II cells are rare (Caicedo et al., 2002; Tomchik et al., 2007; Yoshida, Miyauchi et al., 2009). A combination of several factors could explain the overall higher percentage of type II cells responsive to NaCl and/or KCl that we observed. The most obvious explanation would be that some of the NaCl and/or KCl responses we observed in type II cells were mediated by channels/receptors normally localized to the basolateral membrane of taste cells. In the intact taste bud, these channels would be protected from exposure to most apically applied stimuli by tight junctions that form just below the taste pore (Dando et al., 2015). One such channel could be the hexameric gap junction hemichannel formed by calcium homeostasis modulator 1 and 3 (CALHM1 and CALHM3), which is voltage sensitive and calcium permeable (Ma et al., 2018). CALHM1/CALHM3 is typically associated with mediating adenosine triphosphate (ATP) release following activation of taste GPCRs in type II cells. However, CALHM1-mediated calcium transients have been observed in some type II cells following depolarization (Romanov et al., 2018). Typically, these transients are limited to the microdomains created by the close apposition of atypical mitochondria and the plasma membrane where CALHM1 is localized. However, disruption of these structures, which could have occurred during our cell isolation procedure, can result in more global calcium signals.

Some of the KCl responses in type II cells may have been osmotically driven (Lewandowski et al., 2016), particularly in the case of the 200 mM NaCl stimulus. It is also possible that some of the cells identified as type II based on GFP expression were not yet fully differentiated. This possibility is supported by the fact that 11 of the 40 cells immunopositive for NCAM, a type III cell marker, also expressed Tas1r3, a type II cell marker; this pattern of co-expression has been previously linked to immature taste cells (Miura et al., 2005). Such immature taste cells may be capable of responding to a broader range of stimuli than fully differentiated taste cells. Alternatively, genetic drift in the transgenic mouse lines could have resulted in atypical channel expression in type II cells that enabled responses to salt stimuli not typically seen in C57BL/6 mice. One study using next generation RNA-seq detected expression of Cav2.1 (Cacna1a), the pore forming subunit of P/Q type calcium channels, in Tas1r3-GFP cells (Sukumaran et al., 2017). Previous studies did not detect Cav2.1 in type II taste cells (DeFazio et al., 2006; Roberts et al., 2009). It is not clear if this expression resulted in the production of functional calcium channels in the sequenced Tas1r3-GFP cells because expression of the non-pore forming subunits (alpha-2, beta, and delta) typically associated with P/Q type calcium channels was not detected. Our Tas1r3-GFP cell data came from mice separated from those in the RNA-seq study by several rounds of C57BL/6 backcrossing. Finally, it is possible that intact taste bud recordings underreport NaCl responses in taste cells. It is difficult to explain why this would be the case; however, it is worth noting that recordings from intact taste buds find that salt stimuli generally elicit weaker responses and activate fewer taste cells than sweet, bitter, umami, or sour stimuli (Caicedo et al., 2002; Tomchik et al., 2007; Yoshida, Miyauchi et al., 2009). These results would seem at odds with the strength of gustatory nerve responses to NaCl and with the fact that salty taste is at least as perceptually distinct and intense as the other basic taste qualities. That said, there are numerous mechanisms by which the relatively weak NaCl responses observed at the level of the taste bud could be amplified on their way from the periphery.

We observed spilanthol enhancement in just over 50% of NaCl-responsive type II cells. If, as has been hypothesized for other cell types, spilanthol causes subthreshold depolarization in type II cells, then it could facilitate activation of the voltage-gated sodium channels expressed by type II cells (Gao et al., 2009; Bigiani, 2016). Although type II cells do not express voltage-gated calcium channels, the number of voltage-gated sodium channel mediated action potentials fired in response to a stimulus has been correlated with the magnitude of ATP release by type II cells (Murata et al., 2010; Medler et al., 2003; Clapp et al., 2006). Known taste transduction pathways in type II cells use G protein signaling and calcium release from intracellular stores. Spilanthol could theoretically potentiate the latter stages of G protein signaling in type II cells where a combination calcium release from intracellular stores and subsequent depolarization result in the opening of CALHM1 channels and release of ATP (Taruno, Matsumoto et al., 2013; Taruno, Vingtdeux et al., 2013; Liman et al., 2014). This could in turn increase the positive feedback from autocrine activation of ATP channels on type II cells resulting in greater release of calcium from intracellular stores (Roper, 2013). However, for this mechanism to enhance responses to NaCl requires the currently unsupported assumption that NaCl can either directly activate G protein signaling in type II cells or indirectly activate transduction pathways downstream of G protein activation. That said, experiments in isolated taste cells found evidence that depolarization alone can elicit ATP release from type II cells (Romanov et al., 2007). So, in theory, if NaCl could directly depolarize type II cells and cause ATP release, a calcium signal could be generated by autocrine activation of ATP receptors that would bypass the taste-related G protein signaling pathway.

The mechanisms proposed for spilanthol-mediated enhancement of NaCl responses may also be capable of enhancing responses to other taste qualities. Although a salty taste enhancer would be particularly beneficial for human health, a nonselective enhancer of taste responses could also have useful applications. Helping to ameliorate hypogeusia caused by certain medications would be one potential application. On the other hand, if spilanthol causes variable levels of enhancement among the different taste qualities, then its usefulness would be more difficult to predict and would likely vary depending on the flavor profile of a given food. The effect of spilanthol on other taste qualities and its actual impact on human taste perception will be important areas for future research.

Conclusions

Spilanthol reduces the response threshold and enhances the response magnitude for NaCl in multiple classes of TBCs, likely through inhibition of K2P channels. Further studies will be needed to help resolve the role that potassium leak currents play in mediating spilanthol’s enhancement of NaCl responses in TBCs. Moreover, the K2P channels sensitive to spilanthol will need to be identified. Psychophysical studies would also be profitable to establish how spilanthol alters the quality of salt sensation in humans. Preliminary data from split-tongue experiments in humans suggest that subthreshold concentrations of spilanthol can increase the perceived saltiness of NaCl (Shoji Y et al., unpublished observations). Thus, data at both the cellular and perceptual level suggest that spilanthol could be an effective and naturally occurring enhancer of salty taste. However, the tingling, buzzing, and numbing sensations elicited by higher concentrations of spilanthol remain an obstacle for its utility as a pure salty taste enhancer, as does the uncertainty regarding whether spilanthol enhances other taste qualities. Use of other unsaturated alkylamides will not help with these difficulties because their somatosensory profile is more extensive than spilanthol. Sanshool and IBA, for example, also elicit cooling sensations suggesting that these compounds may target a somewhat broader range of sensory neural afferents than spilanthol (Bryant and Mezine, 1999; Sugai et al., 2005a, 2005b; Albin and Simons, 2010). Further research will be necessary to determine if spilanthol’s undesirable somatosensory effects can be minimized by, for example, modification of its chemical structure or the development of food preparation techniques that keep oral spilanthol exposure to micromolar levels.

Funding

This work was funded in part by Ogawa & Co. Ltd. as well as Monell Chemical Senses Center and the National Institutes of Health’s National Institute on Deafness and Other Communication Disorders Core Grant P30DC011735. Ogawa Co. Ltd. did not have any influence over the experimental, design or data interpretation, or the decision to publish the data.

Conflicts of Interest

J.X., B.L., K.Y., and B.P.B. declare no conflicts of financial interest. T.M. and Y.S. are employees of Ogawa & Co. Ltd.