No abstract

The transduction of taste is a fundamental process that allows animals to discriminate nutritious from noxious substances. Three taste modalities, bitter, sweet, and amino acid, are mediated by G protein-coupled receptors that signal through a common transduction cascade: activation of phospholipase C ␤2, leading to a breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate, which causes release of Ca 2؉ from intracellular stores. The ion channel, TRPM5, is an essential component of this cascade; however, the mechanism by which it is activated is not known. Here we show that heterologously expressed TRPM5 forms a cation channel that is directly activated by micromolar concentrations of intracellular Ca 2؉ (K1͞2 ‫؍‬ 21 M). Sustained exposure to Ca 2؉ desensitizes TRPM5 channels, but PIP2 reverses desensitization, partially restoring channel activity. Whole-cell TRPM5 currents can be activated by intracellular Ca 2؉ and show strong outward rectification because of voltage-sensitive gating of the channels. TRPM5 channels are nonselective among monovalent cations and not detectably permeable to divalent cations. We propose that the regulation of TRPM5 by Ca 2؉ mediates sensory activation in the taste system. I on channels in the transient receptor potential (TRP) family conduct second messenger-gated currents that play diverse roles in cellular physiology. Many are known to mediate vertebrate and invertebrate sensory transduction, including visual transduction (1), pheromone detection (2-4), thermal reception (5-8), and mechanoreception (9, 10). TRP channels can be divided into three classes: TRPC, TRPV, and TRPM (11). Eight vertebrate TRP channels fall into the TRPM class on the basis of structural similarity to the founding member (melastatin) (12). Some of these channels are activated by varied stimuli, including ADP-ribose (TRPM2) (13,14), Ca 2ϩ (TRPM4) (15), and cold or menthol (TRPM8) (6, 7), whereas others are constitutively active (TRPM7) (16)(17)(18). Recently, a member of this family, TRPM5, was implicated in vertebrate taste transduction (19,20); however, the mechanisms by which it is gated are not known.The transduction of bitter, sweet, and amino acid tastes is mediated by G protein-coupled receptors that are distinct for each modality (reviewed in refs. 21 and 22). Although a variety of mechanisms have been proposed for taste transduction, recent evidence indicates that all three modalities use elements of a common pathway (20, 23); receptors signal through a heterotrimeric G protein to phospholipase C ␤2 (PLC␤2), which breaks down phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), and IP 3 opens Ca 2ϩ -release channels to elevate intracellular Ca 2ϩ . Strong support for this pathway comes from a study of mice that carry a targeted deletion of PLC␤2 and are completely unresponsive to bitter, sweet, and amino acid tastes (20). In addition, it is known that taste cells respond to bitter a...

Five canonical tastes, bitter, sweet, umami (amino acid), salty and sour (acid) are detected by animals as diverse as fruit flies and humans, consistent with a near universal drive to consume fundamental nutrients and to avoid toxins or other harmful compounds. Surprisingly, despite this strong conservation of basic taste qualities between vertebrates and invertebrates, the receptors and signaling mechanisms that mediate taste in each are highly divergent. The identification over the last two decades of receptors and other molecules that mediate taste has led to stunning advances in our understanding of the basic mechanisms of transduction and coding of information by the gustatory systems of vertebrates and invertebrates. In this review, we discuss recent advances in taste research, mainly from the fly and mammalian systems, and we highlight principles that are common across species, despite stark differences in receptor types.

Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.

The transient receptor potential A1 (TRPA1) channel is the molecular target for environmental irritants and pungent chemicals, such as cinnamaldehyde and mustard oil. Extracellular Ca 2؉ is a key regulator of TRPA1 activity, both potentiating and subsequently inactivating it. In this report, we provide evidence that the effect of extracellular Ca 2؉ on these processes is indirect and can be entirely attributed to entry through TRPA1 and subsequent elevation of intracellular calcium. Specifically, we found that in a pore mutant of TRPA1, D918A, in which Ca 2؉ permeability was greatly reduced, extracellular Ca 2؉ produced neither potentiation nor inactivation. Both processes were restored by reducing intracellular Ca 2؉ buffering, which allowed intracellular Ca 2؉ levels to become elevated upon entry through D918A channels. Application of Ca 2؉ to the cytosolic face of excised patches was sufficient to produce both potentiation and inactivation of TRPA1 channels. Moreover, in whole cell recordings, elevation of intracellular Ca 2؉ by UV uncaging of 1-(4,5-dimethoxy-2-nitrophenyl)-EDTA-potentiated TRPA1 currents. In addition, our data show that potentiation and inactivation are independent processes. TRPA1 currents could be inactivated by Mg 2؉ , Ba 2؉ , and Ca 2؉ but potentiated only by Ba 2؉ and Ca 2؉ . Saturating activation by cinnamaldehyde or mustard oil occluded potentiation but did not interfere with inactivation. Last, neither process was affected by mutation of a putative intracellular Ca 2؉ -binding EF-hand motif. In conclusion, we have further clarified the mechanisms of potentiation and inactivation of TRPA1 using the D918A pore mutant, an important tool for investigating the contribution of Ca 2؉ influx through TRPA1 to nociceptive signaling. Members of the transient receptor potential (TRP)2 family of ion channels that are expressed by sensory neurons in dorsal root and trigeminal ganglia serve as sensors for temperature and noxious stimuli (1, 2). Of these, TRPA1 is one of the key sensors for pungent chemicals and environmental irritants and is essential for behavioral responses of mice to conditions that evoke inflammatory pain (3-7). Inflammatory mediators, such as bradykinin, bind to G protein-coupled receptors on nociceptors, initiating a second messenger signaling cascade that leads to Ca 2ϩ influx mediated in part by the opening of Ca 2ϩ -permeable TRPA1 channels (5,8,9). TRPA1 is also activated directly by a wide range of chemicals that cause painful sensations, including food additives, such as mustard oil (MO), cinnamaldehyde (Cin), onion, raw garlic, and thyme; environmental irritants, such as formaldehyde and acrolein (a component of automobile exhaust); and products of oxidative stress (4, 8, 10 -16). Many of these chemicals activate TRPA1 by binding covalently to reactive cysteine residues in the amino terminus of the protein (17, 18), producing a modification of the channel that can last for more than 1 h and which leads to persistent activation of TRPA1 currents (18,19). Ca 2ϩ plays at...