(T1r3)-Expressing Taste Cells

+ Glucose transporters and ATP-gated K (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells

Karen K. Yeea,1, Sunil K. Sukumarana,1, Ramana Kothaa, Timothy A. Gilbertsonb, and Robert F. Margolskeea,2

aMonell Chemical Senses Center, Philadelphia, PA 19104-3308; and bDepartment of Biology, Center for Advanced Nutrition, Utah State University, Logan, UT 84322-5305

Edited* by Linda M. Bartoshuk, University of Florida, Gainesville, FL, and approved February 9, 2011 (received for review January 10, 2011) Although the heteromeric combination of type 1 taste receptors (11). In addition to Sac/Tas1r3, there are multiple quantitative 2 and 3 (T1r2 + T1r3) is well established as the major receptor for trait loci that contribute importantly to sweet taste responses in sugars and noncaloric sweeteners, there is also evidence of T1r- mice (3, 18). Physiological studies with canine lingual epithelium independent sweet taste in mice, particularly so for sugars. Before identified sugar-activated cation currents proposed to function as the molecular cloning of the T1rs, it had been proposed that sweet a sweet receptor (19–21). Plausible candidates to mediate T1r- taste detection depended on (a) activation of sugar-gated cation independent sweet taste include intestinal sugar transporters channels and/or (b) sugar binding to G protein-coupled receptors important in the absorption of dietary carbohydrate (reviewed in + to initiate second-messenger cascades. By either mechanism, sug- refs. 22 and 23) and the ATP-gated K (KATP) channel that serves ars would elicit depolarization of sweet-responsive taste cells, as a metabolic sensor in pancreatic islet cells (reviewed in refs. 24 which would transmit their signal to gustatory afferents. We and 25). Here, we used PCR, in situ hybridization, and immu- examined the nature of T1r-independent sweet taste; our start- nohistochemistry to determine if glucose transporters (GLUTs), ing point was to determine if taste cells express glucose transport- sodium–glucose cotransporters (SGLTs), and KATP subunits ers (GLUTs) and metabolic sensors that serve as sugar sensors in are expressed in taste cells. We also assessed electrophysiologi- other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, cally if functional KATP channels were present in taste cells. Our and immunohistochemistry, we determined that several GLUTs results suggest that these signaling proteins may provide T1r- (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium–glucose cotrans- independent sugar-sensing mechanisms in the sweet-responsive + porter (SGLT1), and two components of the ATP-gated K (KATP) subset of taste cells. metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in Results taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, GLUTs and Metabolic Sensors Are Expressed in Taste Cells. To de- and SUR1 were expressed preferentially in T1r3-positive taste cells. termine if GLUTs known to be present in intestine and/or other Electrophysiological recording determined that nearly 20% of the tissues, or metabolic sensors involved in glucose sensing in the total outward current of mouse fungiform taste cells was com- pancreas might also be present in taste cells, we first examined posed of KATP channels. Because the overwhelming majority of expression of their mRNAs in taste and nontaste (NT) tissues. T1r3-expressing taste cells also express SUR1, and vice versa, it is cDNAs were prepared from taste buds isolated from mouse cir- + likely that KATP channels constitute a major portion of K channels cumvallate (CV) papillae and from lingual epithelial cells devoid in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors of taste cells (negative control); PCR assays were then performed and KATP may serve as mediators of the T1r-independent sweet using primer pairs specific for cDNAs corresponding to GLUT2, taste of sugars. GLUT4, GLUT8, GLUT9B, SGLT1, sulfonylurea receptor (SUR) 1, SUR2A, SUR2B, potassium inwardly rectifying channel gustation | sensory transduction (Kir) 6.1, Kir6.2, and the insulin receptor. By PCR, we observed a higher level of expression of GLUT8, GLUT9B, SGLT1, SUR1, ultiple lines of evidence argue for the heteromeric combi- SUR2A, Kir 6.1, and the insulin receptor in cDNA from taste Mnation of type 1 taste receptors 2 and 3 (T1r2 + T1r3) as tissue than from NT tissue (Fig. 1 A and B). PCR indicated that being the primary sweet taste receptor (reviewed in ref. 1). The mRNAs for GLUT2, GLUT4, SUR2B, and Kir6.2 were not Tas1r3 gene encoding T1r3 is allelic to Sac (2–7), the major ge- expressed in greater amounts in taste vs. NT tissue (Fig. 1 A and netic determinant of saccharin and sugar preference in inbred B). Quantitative evaluation by real-time PCR (qRT-PCR) dem- mice (8–10). KO mice deficient in either Tas1r2 or Tas1r3 display onstrated elevated expression in taste tissue cDNA of GLUT8, profound deficits in their responses to sweeteners (11, 12). Het- GLUT9, SGLT1, SUR1, Kir 6.1, and the insulin receptor (Fig. 1 C erologous expression of the human form of T1r2 + T1r3 generates and D). GLUT4 and SUR2 were not found by qRT-PCR to be receptor activity responsive to a broad array of sweet compounds, more highly expressed in taste tissue cDNA, however (Fig. 1 C including monosaccharide and disaccharide sugars (e.g., glucose, fructose, sucrose), sugar alcohols, small-molecule noncaloric sweeteners (e.g., saccharin, cyclamate, sucralose, aspartame, Author contributions: K.K.Y., S.K.S., R.K., T.A.G., and R.F.M. designed research; K.K.Y., neotame), and large-molecule noncaloric sweeteners (e.g., mon- S.K.S., R.K., and T.A.G. performed research; K.K.Y., S.K.S., T.A.G., and R.F.M. analyzed data; and K.K.Y., S.K.S., T.A.G., and R.F.M. wrote the paper. ellin, thaumatin, brazzein) (7, 13–17). Heterologous expression of Conflict of interest statement: R.F.M. has a personal financial interest in the form of stock the rodent form of T1r2 + T1r3 generates a similarly broad-acting ownership in the Redpoint Bio company and is an inventor on patents and patent appli- receptor but without activity toward the “human-specific sweet- cations that have been licensed to the Redpoint Bio company. No other authors eners” (cyclamate, aspartame, neotame, monellin, and thauma- have conflicts. tin) recognized as sweet by humans and old world primates but not *This Direct Submission article had a prearranged editor. by rodents. 1K.K.Y. and S.K.S. contributed equally to this work. Several studies indicate that there are T1r-independent mech- 2To whom correspondence should be addressed. E-mail: [email protected].

anisms for detecting sugars, however. For example, KO mice This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE lacking T1r3 still respond to multiple sugars, particularly glucose 1073/pnas.1100495108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1100495108 PNAS | March 29, 2011 | vol. 108 | no. 13 | 5431–5436 Downloaded by guest on October 1, 2021 Fig. 2. Expression of sugar transporter and KATP glucose sensor mRNAs in taste cells. In situ hybridization to taste bud-containing tissues from mouse CV and foliate taste papillae was carried out with digoxigenin-labeled RNA probes. Taste-cell hybridization to antisense probes indicated expression in CV and foliate taste cells of GLUT2 (A and C), GLUT4 (E and G), GLUT9 (I and Fig. 1. Expression of sugar transporter, KATP glucose sensor, and insulin K), SGLT1 (M and O), and SUR1 (Q and S). Hybridization to sense probe receptor mRNAs in taste tissue. (A) PCR amplification (35 cycles) of sugar controls (B, D, F, H, J, L, N, P, R, and T) in and around taste cells indicative of transporters (GLUT2, GLUT4, GLUT8, GLUT9b, and SGLT1), gustducin (taste nonspecific background was generally lower than with the corresponding RNA control), and GAPDH (tissue RNA control) from mouse cDNA prepared antisense probes. (Scale bars: 20 μm.) from CV taste tissue and NT lingual epithelial tissue devoid of taste buds. GAPDH is expressed in both cDNA samples, but gustducin is expressed only in the taste cDNA. GLUT8, GLUT9b, and SGLT1 are more highly expressed in available antibodies was done to examine expression in taste cells taste cDNA. cDNAs prepared from tissues known to express these genes fi of GLUTs and KATP glucose sensor subunits. Immunoreactivity to served as positive controls (CTRL) to con rm the ability of primers to amplify GLUT2, GLUT4, SGLT1, SUR1, and Kir 6.1 was observed in the correctly sized product. GUST, gustducin. (B) PCR amplification (35 cycles) mouse taste cells of CV, foliate, and fungiform papillae (Fig. 3). of KATP subunits (SUR1, SUR2A, SUR2B, Kir6.1, and Kir6.2) and insulin re- fl ceptor from mouse cDNA prepared from CV taste tissue and NT lingual Interestingly, much of the SUR1 immuno uorescence appeared epithelial tissue devoid of taste buds. SUR1, SUR2A, Kir6.1, and INSR are to be concentrated within the nucleus of taste cells (Fig. 3 J–L). more highly expressed in taste cDNA. cDNAs prepared from tissues known to The nuclear pattern of staining of SUR1 was confirmed with express these genes served as controls to confirm the ability of primers to DAPI staining (Fig. S2 A–F); higher magnification and triple amplify the correctly sized product. INSR, insulin receptor. (C and D) Taqman staining for SUR1, DAPI, and lamin B indicated that SUR1 was real-time PCR was used to quantitate expression of sugar transporters, KATP internal to the nuclear envelope (Fig. S2 G–M). All primary subunits, and insulin receptor in CV (black bars) taste tissue and NT (gray bars) lingual epithelial tissue devoid of taste buds. Elevated expression in antibodies were validated for their ability to detect appropriate taste cDNA was observed for GLUT8, GLUT9, SGLT1, SUR1, Kir6.1, and insulin immunoreactivity in tissues known to express the proteins of in- receptor. The expression of each gene is plotted as the logarithm of the ratio terest: GLUT2 and SUR1 in pancreas, GLUT4 in skeletal muscle, between its cycle threshold value and that of GAPDH. and SGLT1 in small intestine (Fig. S3 A, B, D, and F). In addition, secondary antibodies were shown to be free of nonspecific immunoreactivity in controls without primary antibodies in pan- and D). For those genes more highly expressed in taste tissue, the creas, skeletal muscle, small intestine, and CV (Fig. S3 C, E, absolute levels of mRNA expression in taste tissue were highest and G–I). for GLUT8, SGLT1, and the insulin receptor. The greatest dif- ferential expression was found with SUR1 and the insulin re- GLUT4, SGLT1, and SUR1 Are Selectively Expressed in T1r3-Positive ceptor, each of which was ∼10-fold more highly expressed in Taste Cells. The data above indicate by multiple independent mRNA from taste tissue than from NT tissue. techniques that GLUTs and KATP subunits are present in taste The cDNA templates for the PCR experiments were derived cells. Were any of these GLUTs or sensors of intracellular glucose from CV taste tissue containing a mixture of taste cells and sur- metabolism to function in taste-cell sensing of glucose, they would rounding epithelial and connective cells or from control NT tissue most likely be found in those taste cells that are known to detect devoid of taste cells. To determine if the mRNAs for these genes glucose and other sweet compounds (i.e., T1r2 + T1r3-positive are selectively expressed in the taste cells themselves, we carried subset of type II taste cells). To examine this possibility, we out in situ hybridization with antisense and sense (control) probes. double-stained taste cells using an antibody against a transporter In situ hybridization to taste bud-containing sections indicated or KATP subunit, along with visualizing the intrinsic fluorescence that mRNAs for GLUT2, GLUT4, GLUT9, SGLT1, and SUR1 of GFP in the T1r3-expressing taste cells from T1r3-GFP trans- are selectively expressed in mouse taste cells in both CV and fo- genic mice (26). In this way, GLUT4, SGLT1, and SUR1 were liate papillae (Fig. 2). In control experiments, each of these probes found to be expressed preferentially in T1r3-positive taste cells was validated with tissue sections known to express these mRNAs: (Fig. 4). Nuclear staining of SUR1, as noted above (Fig. 3 and Fig. GLUT2, GLUT9, and SUR1 in pancreas; GLUT4 in skeletal S2), was again observed in taste cells from foliate and CV papillae muscle; and SGLT1 in small intestine (Fig. S1). Next, indirect (Fig. 4 G and J). Quantitation of taste cells that coexpress GLUT4 immunofluorescent confocal microscopy using commercially or SUR1 with T1r3-GFP (Table 1) determined that (i)90–92% of

5432 | www.pnas.org/cgi/doi/10.1073/pnas.1100495108 Yee et al. Downloaded by guest on October 1, 2021 Fig. 4. Coexpression in taste cells of sugar transporter and SUR1 glucose sensor proteins with T1r3. Indirect immunoﬂuorescence confocal microscopy of taste bud-containing sections from mouse CV and foliate taste papillae was carried out with antibodies against sugar transporters (GLUT4 and

SGLT1) or the KATP subunit (SUR1). Double-staining used intrinsic fluorescence (B, C, E, F, H, I, K, and L) of GFP expressed as a transgene from the T1r3 promoter. Overlaid images indicate frequent coexpression of T1r3 with GLUT4 (A–C), SGLT1 (D–F), and SUR1 (G–L). (Scale bars: 20 μm.) Fig. 3. Expression of sugar transporter and KATP glucose sensor proteins in taste cells. Indirect immunofluorescence confocal microscopy of taste bud- containing sections from mouse CV, foliate, and fungiform taste papillae would expect them to be inhibited by sulfonylurea compounds, fi was carried out with speci c polyclonal antibodies directed against sugar such as glibenclamide, that bind to SUR1. Indeed, glibenclamide transporters (GLUT2, GLUT4, and SGLT1) or KATP subunits (SUR1 and Kir6.1). did inhibit outward (K+) currents in mouse fungiform taste cells Immunofluorescence indicates expression in taste cells of GLUT2 (A–C), GLUT4 (D–F), SGLT1 (G–I), SUR1 (J–L), and Kir6.1 (M–O). [Scale bars: 20 μmfor (Fig. 5). Eighteen (26.9%) of 67 taste cells isolated from the CV and foliate papillae (A, B, D, E, G, H, J, K, M, and N); 10 μm for fungiform fungiform papillae of C57BL/6 mice showed a significant re- papillae (C, F, I, L, and O).] The dashed lines outline the extent of the fun- versible inhibition by 20 μM glibenclamide. On average (n = 18), giform taste buds. 20 μM glibenclamide inhibited 18.70 ± 4.32% of the total outward current in mouse fungiform taste cells, (i.e., KATP channels comprise a significant percentage of total taste-cell K+ channels). – GLUT4-expressing taste cells express T1r3, whereas 13 20% of Given the predominant expression of SUR1 in T1r3 taste cells, an T1r3 cells do not express GLUT4 and (ii)80–85% of SUR1- even higher percentage of glibenclamide-inhibitable KATP chan- expressing taste cells express T1r3, whereas 11–24% of T1r3 cells nels among total K+ channels would likely be found in T1r3- do not express SUR1. Double-staining of GLUT4-expressing expressing taste cells. taste cells with markers for type I (GLAST) or type III (Snap-25) taste cells showed that GLUT4 is not present in either taste-cell Discussion

subtype (Fig. S4). Double-staining of SUR1-expressing taste cells GLUTs, SGLTs, and KATP metabolic sensors play important with markers for type I, type II, and type III cells showed that roles in glucose homeostasis and metabolism throughout the SUR1 is mostly present in type II (TrpM5) taste cells (Fig. S4O) body and in many speciﬁc organs (e.g., gut, pancreas, heart, but not in type I (GLAST) taste cells (Fig. S4F) and that there are skeletal muscle, brain) (reviewed in refs. 22–25). The presence a small number of type III (Snap-25) cells that express SUR1 (Fig. and function in taste cells of these important proteins are largely S4L). Quantitation of CV taste cells that coexpress SUR1 with unknown, however. Before the molecular identiﬁcation of the Snap-25 (Table 1) determined that 17% of SUR1-expressing taste primary sweet taste sensor as a heteromeric combination of two cells express Snap-25. In sum, most SUR1-expressing taste cells family C G protein-coupled receptors (GPCRs), T1r2 + T1r3 are T1r3-expressing type II cells, with the remainder likely being (reviewed in ref. 1), it had been proposed that sweet taste might Snap-25–expressing type III cells. rely on sugar transporters or sugar-gated cation channels (19–21, 27). In revisiting this possibility, we observed the presence in taste Mouse Taste Cells Have Functional KATP Channels. The data above cells of multiple sugar transporters and sugar-sensing molecules show that KATP subunits Kir6.1 and SUR1 are present in taste other than T1rs. By RT-PCR, qRT-PCR, in situ hybridization,

cells, with SUR1 found mostly in the T1r3-expressing taste cells. and immunohistochemistry, we observed that taste cells do in- NEUROSCIENCE Were functional KATP channels present in mouse taste cells, we deed express several GLUTs (GLUT2, GLUT4, GLUT8, and

Yee et al. PNAS | March 29, 2011 | vol. 108 | no. 13 | 5433 Downloaded by guest on October 1, 2021 Table 1. Coexpression of GLUT4 and SUR1 with T1R3 or SNAP25 genetic loss of T1r2, T1r3, or downstream signaling elements in taste cells profoundly affects taste-cell responses to sweet compounds. Gene 2 Heterologous expression of T1r2 + T1r3 generates a receptor responsive to a broad diversity of sweet-tasting compounds, re- Gene 1 GLUT4 SUR1 T1r3 Snap-25 flecting the in vivo response characteristics of the species of origin of the T1rs (7, 13, 15–17). Damak et al. (11) reported that mice Nos. of CV taste cells expressing one or both genes engineered to lack T1r3 are unresponsive to artificial sweeteners — GLUT4 ND 112/124 (90%) ND but still respond well to glucose, as assessed by two-bottle prefer- — SUR1 ND 246/307 (80%) 23/139 (17%) ence tests and chorda tympani nerve recording. In contrast, Zhao — T1r3 112/129 (87%) 246/276 (89%) ND et al. (12) found that behavioral and nerve responses to glucose — Snap-25 ND 23/110 (21%) ND were greatly diminished with a different T1r3 null mouse. These Nos. of foliate taste cells expressing one or both genes authors also observed markedly diminished responses to glucose in — GLUT4 ND 113/123 (92%) T1r2 null mice and no detectable responses to glucose in T1r2/T1r3 — SUR1 ND 51/60 (85%) double-KO mice (12). Zhao et al. (12) concluded that T1r2 and — T1r3 113/141 (80%) 51/67 (76%) T1r3 are the only receptors underlying responses to all sweeteners, Mouse taste cells were doubly stained for GLUT4 and T1r3-GFP, SUR1 and including glucose. The marked differences in responses to glucose T1r3-GFP, or SUR1 and SNAP-25. Singly and doubly labeled cells in the CV of the two different types of T1r3 null mice suggest that there may and foliate taste papillae were then counted. Numerators are the numbers be technical reasons for the inability to observe residual responses of taste cells expressing both gene 1 and gene 2. Denominators are the to glucose in the double-KO mice of Zhao et al. (12), however. numbers of taste cells expressing gene 1. Taste cells expressing both gene Although, heterologously expressed T1r3 alone may respond to 1 and gene 2 as a percentage of those expressing gene 1 are shown in high concentrations of sucrose (12), T1r2 alone does not respond parentheses. ND, not determined. to sugars or sweeteners (7, 13), indicating that signaling proteins other than or in addition to T1r2 underlie T1r3-independent sweet taste responses. Although T1r2 + T1r3 and its downstream sig- GLUT9), SGLT1, components of KATP (SUR1 and Kir 6.1), and the insulin receptor. Consistent with a potential role in sweet naling pathway mediate most sweet taste, including that of glucose taste, GLUT4, SGLT1, and SUR1 were found to be expressed and other sugars, the presence of sweet taste mechanisms inde- selectively or exclusively in T1r3-positive taste cells. This is par- pendent of T1rs has been detected (11, 36). Do GLUTs and SGLT1 expressed in the T1r3-positive subset ticularly noteworthy for SGLT1, given that its appearance outside of taste cells mediate T1r3-independent sweet taste responses of small intestine, kidney, or heart is uncommon (22). Further- and underlie certain unique properties of sugar sweeteners more, a significant portion of the total outward current in mouse inconsistent with T1r-mediated response mechanisms? Earlier fungiform taste cells was composed of sulfonylurea-inhibitable studies (19–21) had identified sugar-activated cation currents and K channels. ATP sugar transport across canine lingual epithelium. Based on these Two other groups have recently examined the presence of physiological studies and several biochemical/binding studies, it GLUTs and K subunits in taste cells. Liu et al. (28) used RT- ATP had been proposed (27) that sweet taste detection uses two dis- PCR and immunohistochemistry to identify SUR1 in rat fungiform a fi tinct pathways: ( ) activation of sugar-gated cation channels and taste cells but did not examine taste-cell type-speci c expression. (b) binding of sugars and sweeteners to GPCRs to initiate second In contrast to our present results with mice, these investigators messenger cascades. Subsequent to the molecular cloning of the found SUR1 to be absent from rat CV taste cells. By laser capture T1rs, a major focus of the field has been on the actions of these microdissection of taste buds and microarray analysis of gene ex- fi receptors and their second messenger pathways. There are some pression, Hevezi et al. (29) identi ed GLUT8, GLUT9, GLUT10, sweet-related phenomena that may better be explained by the GLUT13, SLC2A4RG (a regulator of GLUT4), and the insulin actions of sugar transporters and metabolic sensors, however. For receptor as genes potentially expressed in macaque taste cells/ example, the enhancement of sweet taste by sodium salts (37–39) buds. These authors did not carry out in situ hybridization or im- and the sweet taste of low concentrations of Na+ (40, 41) may be munohistochemistry with any of the GLUTs or the insulin receptor mediated by sodium-dependent glucose uptake into T1r3 taste fi to con rm their expression in taste cells or to determine if they cells via SGLT1. Sensory responses to many artificial sweeteners, in fi are expressed in speci c types of taste cells, however. contrast to responses to sugars, display delayed onsets and offsets Sac The locus is the major genetic determinant of sweet taste (42) and lower maximal sweetness intensity (43). The fast on/off preference and responsiveness to saccharin, sucrose, and other response of carbohydrate sweetness may depend on its rapid up- – Sac sweeteners in mice (8 10). has been shown by multiple take into taste cells via apical transporters, followed by metabo- – Tas1r3 groups (2 7) to be synonymous with , the third member of lism or transport out of taste cells via basolateral sugar transporters. the type I family of taste receptor genes that encodes T1r3. Many The higher peak-magnitude sweetness responses displayed by sug- lines of evidence have established the heteromeric GPCR com- ars in vivo may be explained if sugars act via nonsaturable trans- bination of T1r2 + T1r3 as the predominant receptor underlying porters and saturable T1r2 + T1r3, whereas noncaloric sweeteners sweet taste responses in mice (reviewed in ref. 1). For example, act only on T1r. Consistent with this proposal, it has been observed KO mice lacking T1r2 or T1r3 have profound deficits in sweet that sugars act more broadly than artificial sweeteners in elicit- taste responses (11, 12). In addition, KO mice lacking signaling ing cross-adaptation (i.e., there are sugar-sensing mechanisms proteins downstream of T1r2 + T1r3 (e.g., α-gustducin, phos- used by taste cells that cannot be desensitized by noncaloric sweet- pholipase Cβ2, inositol trisphosphate receptor isotype 3, TrpM5) eners) (44). display markedly reduced or absent responses to sweet taste (as What function might KATP channels play in sweet taste in well as diminished responses to umami and bitter compounds, be- T1r3-containing taste cells? KATP channels in pancreas are cause these signaling proteins also couple to T1r1 + T1r3 and spontaneously active; as β-cell glucose rises and is metabolized, T2rs) (reviewed in ref. 30). In humans, polymorphisms in either the intracellular ratio of ATP to ADP also rises, leading to KATP the Tas1r3 or gustducin (Gnat3) promoter region are associated channel closure, depolarization of the cells, and insulin release. with altered sweet preference (31, 32). Physiological studies show Our recording of glibenclamide-inhibitable K+ currents was that leptin and endocannabinoids affect sweet taste responses via done with a general pool of fungiform taste cells not specifically modulation of the activity or expression levels of T1r3-containing identified as being T1r3-positive or not. Given that the over- sweet receptors (33–35). Thus, physiological regulation of T1r3 or whelming majority of SUR1-expressing taste cells also express

5434 | www.pnas.org/cgi/doi/10.1073/pnas.1100495108 Yee et al. Downloaded by guest on October 1, 2021 + Fig. 5. Functional KATP channels are present in mouse taste cells. (A) Glibenclamide, a sulfonylurea inhibitor of the KATP channel, inhibits outward (K ) currents in a mouse fungiform taste cell. (B) Concentration–response function for glibenclamide inhibition of outward current (at +40 mV) normalized to the inhibition of the current achieved by application of 100 μM glibenclamide. Each data point is from 7 to 16 cells.

T1r3 (and vice versa), it is likely that the sulfonylurea-inhibited metabolic conditions, the tonic activity of KATP channels in T1r3 KATP conductance contributes signiﬁcantly more than 20% of cells would hyperpolarize these taste cells, making it less likely the basal total outward current of T1r3-positive taste cells. KATP that sweetener activation of T1r2 + T1r3 depolarizes the taste channels in T1r3-containing taste cells could provide a robust cell, thereby opposing sweet perception. Conceivably, regulation means to regulate activity of these cells according to the local of T1r3 cell KATP channels may be a physiological means to vary content of metabolizable sweet solutions applied to the tongue or taste-cell sensitivity to sweet compounds according to metabolic the general metabolic state of the organism as communicated to needs. The fact that GLUTs and SGLT1 transporters are coex- taste cells via blood glucose or circulating hormones. If K ATP pressed, along with K channels, in T1r3-positive taste cells channels in T1r3 taste cells are apically disposed, inhibiting this ATP + could lead to transporter uptake of sugars that, when metabo- K current by sulfonylureas or elevated ATP would depolarize these cells. At submaximal levels, the combination of a noncaloric lized, would increase taste-cell ATP levels, inhibit KATP, and sweetener acting via T1r2 + T1r3 to initiate a second-messenger depolarize these taste cells. Furthermore, taste-cell KATP might signaling cascade that depolarizes sweet taste cells, along with a regulate hormone and/or neurotransmitter release from T1r3 caloric sweetener, such as glucose, that, when metabolized, pro- taste cells in response to changes in taste-cell metabolism. Just as β motes closure of T1r3 taste-cell KATP channels, would likely inactivation of pancreatic -cell KATP promotes insulin release, so

provide enhanced perception of sweet taste over that achieved might taste-cell KATP inactivation promote hormone release from NEUROSCIENCE by either sweetener alone. Conversely, under low-glucose/low- taste cells.

Yee et al. PNAS | March 29, 2011 | vol. 108 | no. 13 | 5435 Downloaded by guest on October 1, 2021 The localization of SUR1 in taste cells to the nucleoplasm is (T1r3-GFP) and TrpM5 (TrpM5-GFP) were generated as previously described striking and suggests that it is being targeted by a nuclear lo- (26). Taste and NT RNAs were isolated from WT C57BL/6 mice using a Pure- calization signal or by association with another protein with such Link RNA mini kit from Invitrogen. RT-PCR was done using PCR SuperMix from Invitrogen and primer pairs spanning separate exons. Quantification of a sequence. Previously, SUR1 and functional KATP channels have been observed in the nuclear envelope of pancreatic β cells (45). gene expression was by Taqman Gene Expression using Taqman FAST plates This nuclear localization in T1r3 taste cells could serve to se- (both from Applied Biosystems). RNA probes for in situ hybridization were transcribed from verified full-length cDNA plasmids obtained from com- quester the SUR1 away from the plasma membrane, thereby de- + mercial sources (Open Biosystems or imaGenes). Tissues for in situ hybrid- creasing the KATP component of taste-cell resting K currents or ization were freshly frozen in Tissue-Tek O.C.T. mounting media (Sakura). making them insensitive to glibenclamide and other sulfonylureas. Tissues for immunohistochemistry were fixed in 4% paraformaldehyde/1× Another possibility is that KATP channels in the nucleoplasm or PBS and cryoprotected in 20% (wt/vol) sucrose/1× PBS before embedding in nuclear envelope affect taste-cell transcription of T1r3 itself or of O.C.T. For electrophysiology, taste buds from the fungiform papillae of WT other taste-signaling components. Such an effect on transcription C57BL/6 mice were isolated by well-established procedures (46). Further could alter sweet taste responses over a prolonged period and detailed methods are provided in SI Materials and Methods. serve to relate taste receptor expression to dietary content. ACKNOWLEDGMENTS. We thank Drs. Danielle Reed, Alexander Bachmanov, Materials and Methods Paul Breslin, and Gary Beauchamp, as well as several members of the laboratory of R.F.M., for critical reading of the manuscript. This research was All experiments were performed under National Institutes of Health supported by a grant from PepsiCo (to R.F.M.) and by National Science guidelines for the care and use of animals in research and approved by the Foundation Grant DBI-0216310 (to Gary Beauchamp) in support of Monell’s Institutional Animal Care and Use Committee of Monell Chemical Senses Confocal Microscopy Facility. The anti–SNAP-25 antibody was a gift from Center or Utah State University. All mice used for this study were in the C57BL/ Dr. Paul Breslin, and the anti-SGLT1 antibody was a gift from Dr. Soraya 6J background. Transgenic mice expressing GFP under promoters for T1r3 Shirazi-Beechey.

1. Bachmanov AA, Beauchamp GK (2007) Taste receptor genes. Annu Rev Nutr 27: 25. Nichols CG (2006) KATP channels as molecular sensors of cellular metabolism. Nature 389–414. 440:470–476. 2. Bachmanov AA, et al. (2001) Positional cloning of the mouse saccharin preference 26. Clapp TR, Medler KF, Damak S, Margolskee RF, Kinnamon SC (2006) Mouse taste cells (Sac) locus. Chem Senses 26:925–933. with G protein-coupled taste receptors lack voltage-gated calcium channels and 3. Kitagawa M, Kusakabe Y, Miura H, Ninomiya Y, Hino A (2001) Molecular genetic SNAP-25. BMC Biol 4:7. identification of a candidate receptor gene for sweet taste. Biochem Biophys Res 27. Simon SA (1991) Sweeteners: Discovery, Molecular Design, and Chemoreception, eds Commun 283:236–242. Walters E, Orthoefer FT, DuBois GE (American Chemical Society, Washington, DC), pp 4. Max M, et al. (2001) Tas1r3, encoding a new candidate taste receptor, is allelic to the 237–250. sweet responsiveness locus Sac. Nat Genet 28:58–63. 28. Liu DX, Liu XM, Zhou LH, Feng XH, Zhang XJ (2010) Expression of sulfonylurea 5. Montmayeur JP, Liberles SD, Matsunami H, Buck LB (2001) A candidate taste receptor receptors in rat taste buds. Acta Histochem 10.1016/j.acthis.2010.04.009. gene near a sweet taste locus. Nat Neurosci 4:492–498. 29. Hevezi P, et al. (2009) Genome-wide analysis of gene expression in primate taste buds fi 6. Sainz E, Korley JN, Battey JF, Sullivan SL (2001) Identi cation of a novel member of reveals links to diverse processes. PLoS ONE 4:e6395. – the T1R family of putative taste receptors. J Neurochem 77:896 903. 30. Kinnamon SC, Margolskee RF (2008) The Senses: A Comprehensive Reference, eds – 7. Nelson G, et al. (2001) Mammalian sweet taste receptors. Cell 106:381 390. Basbaum AI, et al. (Elsevier, Amsterdam), pp 219–236. fl 8. Pelz WE, Whitney G, Smith JC (1973) Genetic in uences on saccharin preference of 31. Fushan AA, Simons CT, Slack JP, Drayna D (2010) Association between common – mice. Physiol Behav 10:263 265. variation in genes encoding sweet taste signaling components and human sucrose – 9. Fuller JL (1974) Single-locus control of saccharin preference in mice. J Hered 65:33 36. perception. Chem Senses 35:579–592. fl 10. Bachmanov AA, et al. (1997) Sucrose consumption in mice: Major in uence of two 32. Fushan AA, Simons CT, Slack JP, Manichaikul A, Drayna D (2009) Allelic polymorphism – genetic loci affecting peripheral sensory responses. Mamm Genome 8:545 548. within the TAS1R3 promoter is associated with human taste sensitivity to sucrose. 11. Damak S, et al. (2003) Detection of sweet and umami taste in the absence of taste Curr Biol 19:1288–1293. receptor T1r3. Science 301:850–853. 33. Shigemura N, et al. (2004) Leptin modulates behavioral responses to sweet substances 12. Zhao GQ, et al. (2003) The receptors for mammalian sweet and umami taste. Cell 115: by influencing peripheral taste structures. Endocrinology 145:839–847. 255–266. 34. Yoshida R, et al. (2010) Endocannabinoids selectively enhance sweet taste. Proc Natl 13. Li X, et al. (2002) Human receptors for sweet and umami taste. Proc Natl Acad Sci USA Acad Sci USA 107:935–939. 99:4692–4696. 35. Jyotaki M, Shigemura N, Ninomiya Y (2010) Modulation of sweet taste sensitivity by 14. Xu H, et al. (2004) Different functional roles of T1R subunits in the heteromeric taste orexigenic and anorexigenic factors. Endocr J 57:467–475. receptors. Proc Natl Acad Sci USA 101:14258–14263. 36. Delay ER, Hernandez NP, Bromley K, Margolskee RF (2006) Sucrose and monosodium 15. Jiang P, et al. (2005) Lactisole interacts with the transmembrane domains of human glutamate taste thresholds and discrimination ability of T1R3 knockout mice. Chem T1R3 to inhibit sweet taste. J Biol Chem 280:15238–15246. Senses 31:351–357. 16. Jiang P, et al. (2004) The cysteine-rich region of T1R3 determines responses to 37. Bartoshuk LM, Harned MA, Parks LH (1971) Taste of water in the cat: Effects on intensely sweet proteins. J Biol Chem 279:45068–45075. sucrose preference. Science 171:699–701. 17. Winnig M, Bufe B, Meyerhof W (2005) Valine 738 and lysine 735 in the fifth 38. Kumazawa T, Kurihara K (1990) Large enhancement of canine taste responses to transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat – sweet taste receptor. BMC Neurosci 6:22. sugars by salts. J Gen Physiol 95:1007 1018. 18. Reed DR, Bachmanov AA, Beauchamp GK, Tordoff MG, Price RA (1997) Heritable 39. Ugawa T, Konosu S, Kurihara K (1992) Enhancing effects of NaCl and Na phosphate – variation in food preferences and their contribution to obesity. Behav Genet 27: on human gustatory responses to amino acids. Chem Senses 17:811 815. 373–387. 40. Bartoshuk LM, McBurney DH, Pfaffmann C (1964) Taste of sodium chloride solutions “ ” 19. Desimone JA, Heck GL, Mierson S, Desimone SK (1984) The active ion transport after adaptation to sodium chloride: Implications for the water taste . Science 143: – properties of canine lingual epithelia in vitro. Implications for gustatory transduction. 967 968. J Gen Physiol 83:633–656. 41. Bartoshuk LM, Murphy C, Cleveland CT (1978) Sweet taste of dilute NaCl: – 20. Mierson S, DeSimone SK, Heck GL, DeSimone JA (1988) Sugar-activated ion transport Psychophysical evidence for a sweet stimulus. Physiol Behav 21:609 613. in canine lingual epithelium. Implications for sugar taste transduction. J Gen Physiol 42. DuBois GE, Lee JF (1983) A simple technique for the evaluation of temporal taste 92:87–111. properties. Chem Senses 7:237–247. 21. Simon SA, Labarca P, Robb R (1989) Activation by saccharides of a cation-selective 43. DuBois GE, et al. (1991) Sweeteners: Discovery, Molecular Design, and Chemoreception, pathway on canine lingual epithelium. Am J Physiol 256:R394–R402. eds Walters DE, Orthoefer FT, DuBois GE (American Chemical Society, Washington, DC), 22. Scheepers A, Joost HG, Schürmann A (2004) The glucose transporter families SGLT and pp 261–276. GLUT: Molecular basis of normal and aberrant function. JPEN J Parenter Enteral Nutr 44. Schiffman SS, Cahn H, Lindley MG (1981) Multiple receptor sites mediate sweetness: 28:364–371. Evidence from cross adaptation. Pharmacol Biochem Behav 15:377–388. 23. Zhao FQ, Keating AF (2007) Functional properties and genomics of glucose 45. Quesada I, et al. (2002) Nuclear KATP channels trigger nuclear Ca(2+) transients that transporters. Curr Genomics 8:113–128. modulate nuclear function. Proc Natl Acad Sci USA 99:9544–9549. 24. McTaggart JS, Clark RH, Ashcroft FM (2010) The role of the KATP channel in glucose 46. Baquero AF, Gilbertson TA (2010) Insulin activates epithelial sodium channel (ENaC) homeostasis in health and disease: More than meets the islet. J Physiol 588: via phosphoinositide 3-kinase in mammalian taste receptor cells. Am J Physiol 3201–3209. 10.1152/ajpcell.00318.2010.

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