Cellular/MolecularThe Transduction Channel TRPM5 Is Gated by IntracellularCalcium in Taste CellsZheng Zhang,1Zhen Zhao,1Robert Margolskee,2and Emily Liman11Department of Biological Sciences and Program in Neuroscience, University of Southern California, Los Angeles, California 90089, and2Department ofNeuroscience, Mount Sinai School of Medicine, New York, New York 10029Bitter, sweet, and umami tastants are detected by G-protein-coupled receptors that signal through a common second-messenger cascadeinvolving gustducin, phospholipase C␤2, and the transient receptor potential M5 (TRPM5) ion channel. The mechanism by whichphosphoinositide signaling activates TRPM5 has been studied in heterologous cell types with contradictory results. To resolve this issueand understand the role of TRPM5 in taste signaling, we took advantage of mice in which the TRPM5 promoter drives expression of greenfluorescent protein and mice that carry a targeted deletion of the TRPM5 gene to unequivocally identify TRPM5-dependent currents intaste receptor cells. Our results show that brief elevation of intracellular inositol trisphosphate or Ca2⫹is sufficient to gate TRPM5-dependent currents in intact taste cells, but only intracellular Ca2⫹is able to activate TRPM5-dependent currents in excised patches.Detailed study in excised patches showed that TRPM5 forms a nonselective cation channel that is half-activated by 8␮M Ca2⫹and thatdesensitizes in response to prolonged exposure to intracellular Ca2⫹. In addition to channels encoded by the TRPM5 gene, we found thattaste cells have a second type of Ca2⫹-activated nonselective cation channel that is less sensitive to intracellular Ca2⫹. These dataconstrain proposed models for taste transduction and suggest a link between receptor signaling and membrane potential in taste cells.Key words: transient receptor potential; bitter; sweet; inositol trisphosphate; ion channel; calciumIntroductionTaste receptor cells are modified epithelial cells that transducesensory input into changes in the release of neurotransmitteronto afferent nerve fibers. For bitter, sweet, and umami tastants,stimuli bind to seven transmembrane receptors on the apicalsurface of the cells, where they initiate a second-messenger sig-naling cascade, in which phosphoinositide signaling plays a crit-ical role (Margolskee, 2002; Montmayeur and Matsunami, 2002;Medler and Kinnamon, 2004). Exposure of taste tissue to bitterand sweet tastants activates phospholipase C (PLC), leading tohydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2)todi-acylglycerol (DAG) and inositol trisphosphate (IP3) (Hwang etal., 1990; Bernhardt et al., 1996; Spielman et al., 1996; Rossler etal., 1998; Huang et al., 1999). More direct evidence for the im-portance of phosphoinositide signaling in taste transductioncomes from the observation that mice with a targeted deletion ofthe PLC␤2 gene have diminished sensitivity to bitter, sweet, andumami tastants (Zhang et al., 2003; Dotson et al., 2005). Thetransient receptor potential M5 (TRPM5) ion channel, which isspecifically expressed in taste cells (Perez et al., 2002), is criticalfor this signaling cascade, and mice with a targeted deletion ofTRPM5 have little or no ability to detect physiologically relevantconcentrations of bitter or sweet substances (Zhang et al., 2003;Damak et al., 2006). Thus, it can be hypothesized that taste trans-duction involves a PLC signaling cascade that leads to activationof the TRPM5 ion channel, a signaling pathway that is similar, inmany respects, to phototransduction in the fly eye (Montell andRubin, 1989; Ranganathan et al., 1995; Hardie and Raghu, 2001).How does PLC signaling regulate the activity of TRPM5 ionchannels? This question has been addressed by studying the prop-erties of cloned TRPM5 channels expressed in heterologous cellstype with contradictory results. In particular, although one studyshowed that activation of rat TRPM5 channels was independentof an elevation of intracellular IP3or the ensuing rise in intracel-lular Ca2⫹(Zhang et al., 2003), other studies showed that mouseTRPM5 channels could be directly activated by intracellularCa2⫹(Hofmann et al., 2003; Liu and Liman, 2003; Prawitt et al.,2003) or by Ca2⫹store depletion (Perez et al., 2002). To add tothe uncertainty concerning the mechanism by which TRPM5channels are activated, it is likely that heterologously expressedchannels do not fully recapitulate the properties of native chan-nels, which may be composed of multiple subunits (Chen et al.,1993).To directly determine the mechanism by which native TRPM5channels are activated, we measured ionic currents in taste recep-tor cells from genetically modified mice, in which TRPM5-dependent currents could be unequivocally identified. Our re-sults show that native TRPM5 channels are directly activated byintracellular Ca2⫹, downstream of IP3-mediated release of Ca2⫹from intracellular stores.Received Nov. 15, 2006; revised April 18, 2007; accepted April 19, 2007.This work was supported by National Institute on Deafness and Other Communication Disorders Grants 004564(E.L.) and 003155 (R.M.). We thank S. Kinnamon, N.Chaudhari,D.Liu,andY.Wangforhelpwithtastecellisolation;D. Arnold, D. McKemy, and R. Chow for helpful discussions; and H. Waters for expert technical support.Correspondence should be addressed to Emily R. Liman, Program in Neurosciences, University of Southern Cali-fornia, 3641 Watt Way, Los Angeles, CA 90089. E-mail: [email protected]:10.1523/JNEUROSCI.4973-06.2007Copyright © 2007 Society for Neuroscience 0270-6474/07/275777-10$15.00/0The Journal of Neuroscience, May 23, 2007 • 27(21):5777–5786 • 5777Materials and MethodsTransgenic mice. Transgenic mice in which 11 kb of mouse Trpm5 5⬘flanking sequence drives expression of enhanced green fluorescent pro-tein (eGFP) (TRPM5–GFP) and mice carrying a targeted deletion of theTRPM5 gene (TRPM5⫺/⫺) were described previously (Clapp et al., 2006;Damak et al., 2006). The two strains of mice were mated to generateanimals that were positive for the GFP reporter and were ⫹/⫹, ⫹/⫺,or⫺/⫺ for TRPM5 expression. Animals were genotyped as described pre-viously (Clapp et al., 2006; Damak et al., 2006). Additional TRPM5⫹/⫹animals were generated by crossing these animals with wild-type mice ofthe B6D2 strain (Harlan, Indianapolis, IN).Antibody generation and immunostaining of taste tissue. The terminal70 amino acids of mouse TRPM5 (amino