Sugar-regulated cation channel formed by an insectgustatory receptorKoji Sato1, Kana Tanaka1, and Kazushige Touhara2

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan

Edited by King-Wai Yau, The Johns Hopkins School of Medicine, Baltimore, MD, and approved June 6, 2011 (received for review January 3, 2011)

Insects sense the taste of foods and toxic compounds in theirenvironment through the gustatory system. Genetic studies usingfruit flies have suggested that putative seven-transmembranegustatory receptors (Grs) expressed in gustatory sensory neuronsare required for responses to specific tastants. We reconstitutedsugar responses of Bombyx mori Gr-9 (BmGr-9), a silkworm Gr, intwo heterologous expression systems. Xenopus oocytes or HEK293Tcells expressing BmGr-9 selectively responded to D-fructose withan influx of extracellular Ca2+ and a nonselective cation currentconductance in a G protein-independent manner. Outside-outpatch-clamp recording of BmGr-9–expressing cell membranes pro-vides evidence supporting the hypothesis that BmGr-9 constitutesa ligand-gated ion channel. The fructose-activated current associ-ated with BmGr-9 was suppressed by other hexoses, includingglucose and sorbose. The activation and inhibition of insect Grion channels may be the molecular basis for the decoding systemthat discriminates subtle differences in sweet taste. Finally, Dro-sophila melanogaster Gr43a (DmGr43a), a BmGr-9 ortholog, alsoresponded to D-fructose, suggesting that DmGr43a relatives ap-pear to compose the family of fructose receptors.

olfactory | odorant receptor | ionotropic | feeding

In insects, taste and the gustatory systems play a critical role inmultiple behaviors, including feeding, toxin avoidance, court-

ship, mating, and oviposition. Gustatory organs are widely dis-tributed over the entire surface of the body, enabling insects toefficiently detect nonvolatile chemosensory information such aspotential foods or toxic compounds. Taste substances are recog-nized by gustatory sensory neurons that express putative seven-transmembrane proteins in the gustatory receptor (Gr) family.Like the odorant receptor (Or) family, the Gr family is encodedby many related but diverse genes, and genome projects haverevealed 68, 13, 76, and 65 Gr genes in the fruit fly (1), honeybee(2), mosquito (3), and silkworm moth (4), respectively.Molecular genetic studies using the fruit fly Drosophila mela-

nogaster support the role of the Gr family in taste perception.DmGr5a is expressed in gustatory organs and is an essential Gr fora sugar trehalose perception (5–7). Multiple Grs have been im-plicated in the detection of sweet (8, 9) and bitter tastes (10–12)and CO2 vapors (13, 14). Genetic studies have suggested that Gproteins are involved in a Gr-mediated signaling cascade (15–18),but recognizable sequence motifs that support this hypothesisare not present in Gr sequences. Thus, the existing evidence hasled to debate about the molecular mechanisms underlying insecttaste perception.Similar to the Gr, insect Or genes also encode proteins with

seven-transmembrane domains, but lack the apparent G protein-coupled receptor motifs (19). Evidence for atypical signaltransduction characteristics of insect Ors came from studies ofsilkmoth Bombyx mori Ors (20), and later it was found that insectOrs had the capacity to act as ligand-gated ion channels directlyactivated by odorants (21, 22). Thus, it seems reasonable topropose that insect Grs may also share an ionotropic couplingmechanism with the insect Ors (23). In this study, we examinedthe detailed molecular response of a single Gr to fructose in

heterologous expression systems and propose that this Gr con-stitutes a nonselective, fructose-activated cation channel.

ResultsD-Fructose Is a Specific Ligand for a B. mori Gustatory Receptor,BmGr-9. We examined 14 of the 65 B. mori Grs (BmGr) that weremost closely related to Gr genes in other insects. RT-PCRexperiments demonstrated that 11 of these 14 BmGrs were tran-scribed in gustatory organs (Fig. S1); 8 of the 11 that were func-tionalGrs (3 were pseudogenes) were expressed in Xenopus laevisoocytes for functional analysis. Using the two-electrode voltage-clamp technique, oocytes injected withGr cRNA were stimulatedwith tastants, including compounds that have been reported toelicit feeding behavior or electrical responses in silkworms (24). Ata holding potential of –80 mV, an inward current was recordedfrom oocytes expressing BmGr-9 in response to D-fructose,a monosaccharide hexose (Fig. 1A). On the basis of the dose–response curve, the EC50 value of D-fructose was 56 mM forBmGr-9 (Fig. 1B). The threshold concentration (∼0.3 mM) wasslightly lower than that necessary for an electrophysiological re-sponse to D-fructose in silkworms in vivo (5–10mM) (24). BmGr-9recognized D-fructose very selectively and precisely discriminatedbetweenmolecules (e.g., an enantiomer and stereoisomers) on thebasis of the stereo-configuration of a hydroxyl group on the hexosering (Fig. 1A).Interestingly, there were dose-dependent changes in current-

voltage (I–V) relationships; at near-threshold concentrations ofD-fructose, the current showed strong inward rectification (Fig.1C, Left), whereas the rectification was abolished at concen-trations over the EC50 without a shift in reversal potential (Fig.1C, Right). These results suggested that D-fructose was a ligandfor BmGr-9 and that activation of BmGr-9 leads to the gener-ation of a depolarizing receptor potential in oocytes.We also expressed BmGr-9 in HEK293T cells and performed

Ca2+ imaging. HEK293T cells expressing BmGr-9 exhibited Ca2+

responses to D-fructose in a dose-dependent manner with anEC50 value of 35 mM and a threshold concentration of 3 mM(Fig. 1 D and E). A specific response to D-fructose was also ob-served in the Ca2+ imaging experiment (Fig. S2).BmGr-9 belongs to one of the functionally unknown members

of the insect Gr family that forms a highly confident single lin-eage on the phylogenetic tree (4). We then tested the function ofDrosophila melanogaster Gr43a (DmGr43a), a BmGr-9 ortholog.

Author contributions: K.S., K. Tanaka, and K. Touhara designed research; K.S. andK. Tanaka performed research; K.S., K. Tanaka, and K. Touhara analyzed data; and K.S.and K. Touhara wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database (accession nos. AB600835, AB600836, and AB600837).1K.S. and K. Tanaka contributed equally to this work.2To whom correspondence should be addressed. E-mail: ktouhara@mail.ecc.u-tokyo.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019622108/-/DCSupplemental.

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COS-7 cells expressing DmGr43a exhibited Ca2+ responses toD-fructose in a dose-dependent manner (Fig. 1F).

Characterization of D-Fructose–Stimulated Ca2+ and Electric Signalsin BmGr-9–Expressing HEK293T Cells. We next characterized thesource of increase in intracellular Ca2+ when BmGr-9–express-ing cells are stimulated by D-fructose. Chelating extracellular Ca2+

with EGTA eliminated the D-fructose–activated Ca2+ increase,indicating that the Ca2+ increase resulted from the influx of ex-tracellular Ca2+ (Fig. 2 A and B). The basal Ca2+ levels in BmGr-9–expressing cells significantly decreased in the presenceofEGTA(Fig. 2 A and B), suggesting that BmGr-9 mediates a spontaneousCa2+ influx in a manner similar to an insect Or complex (21).We performed pharmacological experiments to determine

whether the D-fructose–evoked Ca2+ influx required a G protein-mediated intracellular signaling cascade. U73122, an inhibitorof phospholipase C, abolished α1-adrenergic receptor-mediated

Ca2+ responses, but did not affect the BmGr-9–evoked Ca2+

responses (Fig. 2C). Although eugenol stimulation resulted incAMP production in HEK293T cells expressing mouse eugenololfactory receptor (mOR-EG), no cAMP increase resulted fromD-fructose stimulation of BmGr-9–expressing HEK293T cells(Fig. 2D). Application of a cell membrane-permeable cyclic-nucleotide analog (either 8-bromo-cGMP or 8-bromo-cAMP) oran adenylyl cyclase activator (forskolin) failed to produce Ca2+

responses in BmGr-9–expressing HEK293T cells (Fig. 2E).To examine whether G protein signaling was necessary for

BmGr-9 activation more directly, we loaded 2 mM GDP-βS,a non-hydrolyzable form of GDP that inhibits G protein-coupledsignaling, into the HEK293T cells and performed whole-cellpatch-clamp experiments. GDP-βS significantly inhibited endo-geneous muscarinic receptor-mediated current responses in-duced by carbachol, but GDP-βS had no effect on the currentresponse of BmGr-9 to D-fructose (Fig. 2F). These results sug-

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Fig. 1. D-Fructose is a ligand for a B. mori gustatory receptor, BmGr-9, and a D. melanogaster gustatory receptor, DmGr43a. (A) The current traces recordedfrom BmGr-9–expressing Xenopus oocytes or control oocyte (no injection) with sequential application of various sweeteners at the holding potential of −80mV. Tastants were applied for 3 s at the time indicated by arrowheads. All compounds were tested at 100 mM. The data are representative of recordings fromten oocytes. (B) The BmGr-9 current was dependant on the dose of D-fructose. (Upper) The current responded to application of the indicated concentrationsof D-fructose. D-Fructose was sequentially applied for 3 s to the same oocyte at the time points indicated by the red arrowheads. (Lower) Dose–response curveof BmGr-9. The curve was fitted to the Hill equation (n = 6; EC50 = 55.5 mM, Hill coefficient = 1.02). (C) Current-voltage (I–V) relationships resulting fromtreatments with several concentrations of D-fructose. The data are representative of recordings from four oocytes. (D) Dose-dependent Ca2+ responses ofHEK293T cells expressing BmGr-9 to D-fructose. The pseudocolored images demonstrate the changes in Fura-2 fluorescence with increasing concentrationsof D-fructose where red indicates a cell that shows the greatest response. The chart shows average Fura-2–based Ca2+ responses to increasing concentrationsof D-fructose (n = 34), and the bar indicates the timing of stimulation. (E) Dose–response curve of BmGr-9 to D-fructose based on quantitative analysis of Ca2+

imaging. The curve was fitted to the Hill equation (n = 159; EC50 = 35 mM, Hill coefficient = 1.62). (F) Average Fura-2–based Ca2+ responses of COS-7 cellsexpressing DmGr43a with application of 1 and 10 mM D-fructose (n = 10). The shaded region around the trace represents SEM.

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gest that G protein-signaling cascades are not involved in theresponse of BmGr-9 to D-fructose.

D-Fructose–Activated Cation Channel Activity Produced by BmGr-9.We then hypothesized that BmGr-9 formed a ligand-gated cationchannel, similar to an insect Or complex (21, 22). Inward cur-rents were observed in whole-cell voltage-clamp recordings ofBmGr-9–expressing HEK293T cells at a holding potential of –60mV (Fig. 3A). The average latency of the BmGr-9–mediatedcurrent was 73 ± 6.2 ms, ranging from 22 to 201 ms (n= 35) (Fig.3B). The slopes of the initial BmGr-9 currents were superim-posable regardless of the duration of stimulation (Fig. 3B), un-like the case of G protein-mediated odorant responses invertebrates (21, 25). In a manner similar to the results from theoocyte recordings (Fig. 1C), the BmGr-9 current showed inwardrectification at near-threshold concentrations of D-fructose (Fig.3C). COS-7 cells expressing DmGr43a also exhibited the currentresponses to 100 mM D-fructose without current rectification(Fig. 3D). Reversal potentials changed depending on the cationcomposition in the external and internal solution at 100 mMD-fructose stimulation, suggesting that BmGr-9 elicited a non-selective cation conductance (Fig. 3E). The 100 mM D-fructose–

evoked inward current was inhibited by a calcium channelblocker, ruthenum red (Fig. 3F).We further characterized the source of BmGr-9–evoked cur-

rent induced by D-fructose. The macroscopic currents recordedfrom the outside-out cell membrane of a BmGr-9–expressingHEK293T cell showed electrical characteristics similar to thoseof whole-cell currents (Fig. S3A). Finally, outside-out patch-clamp recordings from HEK293T cell membrane expressingBmGr-9 showed unitary currents of 1.29 ± 0.083 pA [responseindex (RI) ranging from 0.07 to 0.76; n = 5] with a conductanceof 17.1 picosiemens (pS) when cells were held at –60 mV uponstimulation with D-fructose (Fig. 3G). The activation of channelopening by D-fructose was not observed from vector-transfectedcell membranes (n = 10). This measured conductance wasconsistent with a single-channel conductance obtained by anoise analysis (26): 14.9 ± 0.88 pS by the linear fit (n = 4)(Fig. S4). Taken together, these results provide evidence sup-porting the hypothesis that BmGr-9 forms a D-fructose–activatedcation channel.

Inhibition of BmGr-9–Mediated Currents by Hexoses. We next ex-amined whether BmGr-9 channel activity was modulated byother sugars. No enhancement or suppression was observed

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Fig. 2. Characterization of D-fructose–stimulated Ca2+ and electric signals in BmGr-9–expressing HEK293T cells. (A) Average Ca2+ responses of BmGr-9–expressing HEK293T cells (n = 29; Left) or vector (n = 29; Right) to 100 mM D-fructose (20-s stimulation) in the presence or absence of 10 mM EGTA (red bar). (B)Quantification of response amplitudes of D-fructose–stimulated Ca2+ responses in A. (C) Average Ca2+ responses of HEK293T cells expressing BmGr-9 plus α1-adrenergic receptor (AR) to 100 mM D-fructose (20-s stimulation) or to 100 nM norepinephrine (5-s stimulation) before and after application of 10 μM U73122(blue bar) (n = 7). (D) Quantification of cAMP in HEK293T cells expressing mOR-EG or BmGr-9 stimulated with eugenol (1 mM) or various concentrations ofD-fructose (from 1 to 300 mM), respectively (n = 4, mean ± SEM). (E) Average Ca2+ responses of HEK293T cells expressing BmGr-9 to D-fructose (100 mM),8-bromo-cAMP (1 mM), 8-bromo-cGMP (1 mM), and forskolin (5 μM) (20-s application) (n = 18). (F) Effect of GDP-βS on ligand-induced whole-cell currents inHEK 293T cells (each: n = 11–13). (Left panels) Arrowheads indicate the timing of the 20-s carbachol application to vector-transfected HEK293T cells. (Rightpanels) Arrows indicate the timing of the 1-s D-fructose application to BmGr-9–transfected HEK293T cells. Recording was performed in the presence (bluetrace) or absence (green trace) of 2 mM GDP-βS in the electrode. Red bar indicates the timing of GDP-βS application (whole-cell mode configuration). Sig-nificance was assessed by t test or ANOVA and Fisher’s protected least squares difference: *P < 0.05, ***P < 0.001. Error bars and shaded regions aroundthe Ca2+ response traces represent SEM.

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when 300 mM of pentose, disaccharide, or trisaccharide (i.e.,arabinose, sucrose, trehalose, or raffinose) was added to 100 mMD-fructose in BmGr-9–expressing oocytes (Fig. 4A). However,some hexoses, including D-glucose, D-galactose, and L-sorbose,suppressed the D-fructose–evoked inward current in BmGr-9–expressing oocytes (Fig. 4A). The inhibitory effect was dose-dependent (Fig. 4 B and C), and the current recovered with in-creasing amounts of D-fructose, suggesting that the hexosescompeted with D-fructose at the ligand binding site on BmGr-9(Fig. 4D). Similarly, the BmGr-9–mediated D-fructose–dependentcurrent in HEK293T cells was inhibited by D-glucose (Fig. 4E,Lower). Spontaneous BmGr-9–mediated activity was also sup-pressed by D-glucose, and a reversal current response was ob-served (Fig. 4E, Upper). The latency of the D-fructose–evokedcurrent was longer in the presence of D-glucose (Fig. 4F). Themacroscopic current activity of the outside-out cell membrane

of a BmGr-9–expressing HEK293T cell was also inhibited byD-glucose (Fig. S3B). Together, these results demonstrated thatthe BmGr-9 channel is positively and negatively regulated bymonosaccharide hexoses.

DiscussionIn this study, we propose that BmGr-9, a silkworm Gr, con-stitutes a nonselective, fructose-activated cation channel that isinactivated by several hexoses, including glucose, galactose, andsorbose. Although genetic studies using Drosophila have sug-gested that coexpression of multiple Grs is necessary to respondto sugars such as D-glucose, trehalose, and sucrose (8, 9), tobitter compounds such as caffeine and theophylline (10–12), andto CO2 (13, 14), BmGr-9 appears not to require the expression ofother Grs to show the responsiveness to D-fructose in vitro.BmGr-9–expressing neurons, however, may coexpress other Grs

Fig. 3. BmGr-9 forms a D-fructose–activated cation channel. (A) A whole-cell current recorded from a HEK293T cell expressing BmGr-9 upon stimulation with1 or 100 mM D-fructose for 200 ms. (B) Onset of BmGr-9–mediated inward current in response to D-fructose stimulus of increasing duration as indicated by thedifferent colors. (C) Current response of a HEK293T cell expressing BmGr-9 (10 or 100 mM D-fructose at various holding potentials). Top traces indicate onsetof stimulation. (Left) Blue shows a cell with strong inward rectification, and (Right) green shows a cell without rectification. (Right) I–V relationship with therespective peak current of each response in Left panel plotted. (D) A whole-cell current recorded from a COS-7 cell expressing DmGr43a upon stimulation with100 mM D-fructose for 2 s (first application) or 5 s (second and third applications). (Right) I–V relationship. (E) Reversal potentials of whole-cell currentresponses of HEK293T cells expressing BmGr-9 [100 mM D-fructose resulting from different cation compositions in the recording solution (each: n = 7–24)]. Ionand reagents in the external (Ext.) and internal (Int.) solutions are indicated at the bottom. (F) Effect of ruthenium red at a holding potential of +80 and −60mV on the whole-cell current response of a HEK293T cell expressing BmGr-9. The timing of 100 mM D-fructose and 50 μM ruthenium red application is in-dicated by white and red bars, respectively. (G) Excised outside-out patch-clamp recording of BmGr-9 currents measured in a HEK293T cell membrane. The toptrace shows the timing of the addition of 100 mM D-fructose. All-point current histograms (bottom) were obtained from the region indicated on a trace offirst stimulation by the green bar before stimulation (green histogram, left) and by the blue bar during stimulation (blue histogram, right). (Right) Recordingof an untransfected cell membrane. Mean peak current levels were obtained from the fitted Gaussians.

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among 65 BmGr genes, and therefore, we cannot exclude thepossibility that BmGr-9 exhibits different ligand response prop-erties in vivo.BmGr-9 and its orthologs, including DmGr43a, form a distinct

Gr subfamily that is not categorized in the sugar or bitter re-ceptor subfamilies but are conserved within endopterygoteinsects (4). We demonstrated that both BmGr-9 and DmGr43aresponded to D-fructose, suggesting that the DmGr43a orthologfamily may represent a Gr subfamily that plays a role inD-fructose perception. Although the physiological function ofD-fructose in insect species has not been well characterized, thefact that the DmGr43a family is separated from other sugar re-ceptor gene families on the phylogenetic tree indicates thatD-fructose may play a distinct physiological role in insects. In thisregard, it is intriguing to note that BmGr-9 is also expressed inthe gut (Fig. S1), suggesting the involvement in intestinal ab-sorption or some metabolism.As is the case for insect Ors, the insect gustatory system has

long been thought to use a G protein-mediated signaling path-way. Genetic ablation of Gαs, Gαq, Gγ1, or phospholipid sig-

naling resulted in partial reduction in trehalose responses inDrosophila (15, 16, 18). Additionally, Gαo is evidently involved inthe detection of sucrose, glucose, and fructose in Drosophila (27).However, there was no evidence for a second messenger-medi-ated pathway in the BmGr-9 response to D-fructose. Our resultswere consistent with previous electrophysiology experiments thatsuggested the presence of D-fructose-driven ion channel trans-duction in the flesh-fly sugar receptor neurons (28), and thesesugar-activated currents showed nonselective cation conductancein vivo (29).In conclusion, we provide several lines of evidence supporting

the hypothesis that an insect Gr is an ionotropic channel regu-lated by taste substances. Similar to the olfactory system,a channel that is both positively and negatively regulated playsa role in taste perception in insects. Although the insect Ors andGrs do not resemble each other at the amino acid sequence level,our findings suggest that these insect chemosensory systems havea common mechanism for decoding a variety of chemical signalsin the external environment. Our success in reconstituting thesugar responsiveness of an insect Gr and in performing phar-macology in vitro paves the way for characterizing the remaininginsect Grs and for a better understanding of the contribution ofmany Grs to taste perception and the regulation of feedingbehaviors in insects.

Materials and MethodsInsects. Eggs of the silkworm B. mori (hybrid strain, Kinshu × Showa) werepurchased from Ueda Sanshu Ltd.. Larvae were reared in plastic containersat 25 °C with 70% relative humidity and long-day lighting conditions (16 hlight/8 h dark) on a SILKMATE 2S artificial diet (Nippon Nosan Co. Ltd.).Larvae were provided with fresh food on a daily basis, and all larvae werestaged to synchronize growth.

RT-PCR. Total RNAwas isolated from the antennae of 10male adult moths, 10female adult moths, and 10 larvae maxilla; from the labrum, mandible, la-bium, thoracic, and proleg of 10 larvae; and from the gut of one larva usinga Microto-Midi Total RNA Purification System (Invitrogen). Following treat-ment with DNase I (Promega), cDNA was produced using SuperScript III(Invitrogen). cDNA was amplified using Ex Taq DNA polymerase (Takara)under the following reaction conditions: 94 °C for 5 min and then 40 cycles at94 °C for 30 s, * °C for 30 s, and 72 °C for 2 min, followed by 72 °C for 7 min[asterisk (*) indicates BmGr-1, -2, -5, -9, -66, and -67 primers at 60 °C; BmGr-4,-6, -7, -8, -53, and -68 primers at 54 °C; and BmGr-3 and -10 primers at 52 °C].The following primer pairs were used for the PCR: BmGr-3 (5′-ATGTCCTTC-GAAATAAAAAATAATTTC-3′ and 5′-TCAATCATTTTTTCTTTTCGCAAAAGC-3′);BmGr-2 (5′-ATGATTCCGGACCATCTTTTTGAAG-3′ and 5′-TTATTGACCGGT-GCCATGTG-3′); BmGr-1 (5′-ATGAACAGACACGACCATAGATTC-3′ and 5′-TCATTCTTGATCTTCATCACTTCC-3′); BmGr-66 (5′-ATGAAACGTAAATTAAAG-AAGTTTTTTCCG-3′ and 5′-TTACGAACGACGTTGTTCTTG-3′); BmGr-67 (5′-A-TGAGAGAAAGAAAAAAAAAATTTAACAAA-3′ and 5′-TTATCGCCCGCGTC-3′);BmGr-4 (5′-ATGTCACGGATCTTCTCGATG-3′ and 5′-TTATATAGGTACCTCTA-ACAACAATTC-3′); BmGr-53 (5′-ATGGCTCAAATAAAAGATGAAAATCAATC-3′and 5′-TTAGACAAAATGAGAGAGTTGAATAATC-3′); BmGr-8 (5′-ATGGCT-CCTCGATCAGTTC-3′ and 5′-TTAAATTTGAAGTAATACTATTTCGTACGT-3′);BmGr-9 (5′-ATGCCTCCTTCGCCAG-3′ and 5′-TTAACTATCATATCGCTGGAATT-GAATG-3′); BmGr-5 (5′-ATGAGTAAAATTCTAAAATTCCTGAC-3′ and 5′-TTAATCTTCATTGCTGAATTGAAGC-3′); BmGr-6 (5′-ATGGTAACACAGTTCC-TTAACATTC-3′ and 5′-CTACGAGTAGTTGTAAAATGTTTC-3′); BmGr-10 (5′-ATGATCAAAATTCGACTAAAAACATTAAG-3′ and 5′-TTATTGCATATTTTTAA-TTTCCAGCTG-3′); BmGr-7 (5′-ATGTGTTGTTTGGGAGAAACCAG-3′ and 5′-TCATATAAGATGTTTCGGCAAATAATAATC-3′); and BmGr-68 (5′-ATGCGTTT-CGGTTTGAAGGC-3′ and 5′-TTAATTTCTTTTATCAAACTGAACTAAAAT-3′).

Patch-Clamp Experiments in Mammalian Cell Lines and Outside-Out CurrentData Analysis. A full-length cDNA for BmGr-9 and DmGr43awere cloned intothe pME18S vector. This Gr expression vector was transiently transfected intoCOS-7, HeLa, or HEK293T cells with lipofectamine 2000 reagent (Invitrogen).GFP or monomeric red fluorescent protein were cotransfected as a control.Whole-cell currents were amplified with a patch-clamp amplifier (Axopatch200B; Molecular Devices) and were digitized with PowerLab (AD Instru-ments). The extracellular buffer solution contained (in mM) 140 NaCl, 5.6KCl, 5 Hepes, 2.0 pyruvic acid sodium salt, 1.25 KH2PO4, 2.0 CaCl2, and 2.0

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Fig. 4. Inhibition of BmGr-9–mediated currents by hexoses. (A) The D-fruc-tose–evoked whole-cell currents recorded from BmGr-9–expressing Xenopusoocytes were inhibited by coapplication of 300 mM sweeteners (n = 6,mean ± SEM). (B) Dose-dependent inhibition of D-fructose–evoked inwardcurrents in BmGr-9–expressing Xenopus oocytes by D-glucose, D-galactose,or L-sorbose. Current traces upon 3 s application of a mixture of 10 mMD-fructose and the indicated concentration of D-glucose (magenta trace),D-galactose (green trace), or L-sorbose (blue trace); the inhibitors were addedat the time points indicated by the arrowheads. (C) Quantification of dose-dependent inhibition (n = 7, mean ± SEM). (D) The inhibition associated with300 mM D-glucose or L-sorbose can be overcome in BmGr-9–expressingoocytes by increasing the concentration of D-fructose to 30 mM (blue bar) or100 mM (brown bar) (n = 4, mean ± SEM). (E) Current traces of a BmGr-9–expressing HEK293T cell upon application of 100 mM D-fructose (open bar)or 300 mM D-glucose (magenta bar) (F) Onset of inward current in a BmGr-9–expressing HEK293T cell to D-fructose in the presence (magenta trace) orabsence (black trace) of 300 mM D-glucose; the competitor was added at thetime point indicated by the open bar. Significance was assessed by the t test:*P < 0.05, **P < 0.01, ***P < 0.001. The holding potential was −80 mV(Xenopus oocytes) and −60 mV (HEK293T).

11684 | www.pnas.org/cgi/doi/10.1073/pnas.1019622108 Sato et al.

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MgCl2, (pH 7.4). The electrode solution contained (in mM: 140 KCl, 10 Hepes,5 EGTA-2K; pH 7.4). To record the shift in reversal potential and in equilib-rium potential for a specific ion, the following external (bath) and internal(electrode) solutions were used: N-methyl-D-glutamine (NMDG) plus Ca2+

external solution (in mM: 190 NMDG, 40 Hepes, 5.6 KCl, 2.0 pyruvic acidsodium salt, 1.25 KH2PO4, 2.0 CaCl2, and 2.0 MgCl2; pH 7.4); NMDG externalsolution (in mM: 190 NMDG, 40 Hepes, 5.6 KCl, 2.0 pyruvic acid sodium salt,1.25 KH2PO4, and 2.0 MgCl2; pH 7.4); K+plus Ca2+ external solution (in mM:145 KCl, 5 Hepes, 2.0 pyruvic acid sodium salt, 1.25 KH2PO4, 2.0 CaCl2, and 2.0MgCl2; pH 7.4); and Na+ internal solution (in mM: 140 NaCl, 10 Hepes, and 5EGTA-2Na, pH 7.4). For the outside-out recording, both the external andelectrode sodium solutions contained (in mM) 140 NaCl, 5 Hepes, and 2.0pyruvic acid sodium salt, pH 7.4. The data were sampled at 20 kHz and low-pass-filtered at 2 kHz.

For the analysis of an outside-out current recording, the channel con-ductance was first determined by the peak of fitted multiple Gaussianfunction. Next, the responsiveness of a channel was assessed by the RI thatwas defined as the inverted ratio of D-fructose–activated channel activity tobaseline: RI = integration of currents during [t = −10, t = 0] (pA·s)/integrationof currents during [t = 0, t = 10] (pA·s), where t = −10, t = 0, and t = 10correspond to 10 s before D-fructose stimulation, onset of stimulation, and10 s after stimulation, respectively. The values of RI < 1 and RI > 1 wereexpected to obtain upon channel activation and inhibition, respectively. Theresponsiveness of each cell membrane was confirmed by reproducibleresponses to repeated stimulations.

Xenopus Oocyte Electrophysiology. Oocytes were microinjected with 50 ng ofBmGr-9 cRNA. Injected oocytes were then incubated for 3 d at 18 °C in Barth’s

solution [(in mM): 88 NaCl, 1 KCl, 0.3 Ca(NO3) 2, 0.4 CaCl2, 0.8 MgSO4, 2.4NaHCO3, and 15 Hepes, pH 7.6, supplemented with 10 μg/mL penicillin andstreptomycin]. Whole-cell currents were recorded with a two-electrodevoltage clamp filled with 3 M KCl and were amplified with an OC-725Camplifier (Warner Instruments), low-pass-filtered at 50 Hz and digitized at1 kHz. Tastants were applied through the superfusing bath solution [(in mM):115 NaCl, 2.5 KCl, 1.8 MgSO4, 2.4 NaHCO3, and 10 Hepes; pH 7.2]. Solutionswere switched using handmade, Peripheral Interface Controller-driven(16F877, Microchip Technology Inc.) solenoid valves (Cole Parmer).

Ca2+ Imaging. BmGr-9 or DmGr43a was transfected into HEK293T or COS-7cells, which were loaded with 2.5 μM Fura-2/AM for 30 min. Fluorescence wasmeasured with an Aquacosmos Ca2+ imaging system (Hamamatsu Photonics).Tastants and drugs were delivered through the superfusing exracellularbuffer solution.

cAMP Assay. HEK293T cells transfected with BmGr-9 were incubated with1 mM 3-isobutyl-1-methylxanthine (IBMX) for 30 min. The cells were thenexposed to the indicated concentration of D-fructose for 15 min. cAMP levelswere determined with an ELISA kit (Applied Biosystems) in accordance withthe manufacturer’s directions.

ACKNOWLEDGMENTS. We thank M. Tominaga, Y. Okamura, L. B. Vosshall,M. Suwa, and Y. Ono for helpful discussion and criticisms. This work wassupported in part by grants from the Ministry of Education, Culture, Sports,Science and Technology of Japan to K.S. (Grants-in-Aid for ScientificResearch Young Scientist A) and K. Touhara (Grants-in-Aid for ScientificResearch on Priority Areas).

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