Maarten Gees

The Role of Transient Receptor Potential Cation Channels in Ca2+ Signaling

The 28 mammalian members of the super-family of transient receptor potential (TRP) channels are cation channels, mostly permeable to both monovalent and divalent cations, and can be subdivided into six main subfamilies: the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and the TRPA (ankyrin) groups. TRP channels are widely expressed in a large number of different tissues and cell types, and their biological roles appear to be equally diverse. In general, considered as polymodal cell sensors, they play a much more diverse role than anticipated. Functionally, TRP channels, when activated, cause cell depolarization, which may trigger a plethora of voltage-dependent ion channels. Upon stimulation, Ca2+ permeable TRP channels generate changes in the intracellular Ca2+ concentration, [Ca2+]i, by Ca2+ entry via the plasma membrane. However, more and more evidence is arising that TRP channels are also located in intracellular organelles and serve as intracellular Ca2+ release channels. This review focuses on three major tasks of TRP channels: (1) the function of TRP channels as Ca2+ entry channels; (2) the electrogenic actions of TRPs; and (3) TRPs as Ca2+ release channels in intracellular organelles.

Nicotine activates the chemosensory cation channel TRPA1.

Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

Oxaliplatin elicits mechanical and cold allodynia in rodents via TRPA1 receptor stimulation

&NA; Platinum‐based anticancer drugs cause neurotoxicity. In particular, oxaliplatin produces early‐developing, painful, and cold‐exacerbated paresthesias. However, the mechanism underlying these bothersome and dose‐limiting adverse effects is unknown. We hypothesized that the transient receptor potential ankyrin 1 (TRPA1), a cation channel activated by oxidative stress and cold temperature, contributes to mechanical and cold hypersensitivity caused by oxaliplatin and cisplatin. Oxaliplatin and cisplatin evoked glutathione‐sensitive relaxation, mediated by TRPA1 stimulation and the release of calcitonin gene‐related peptide from sensory nerve terminals in isolated guinea pig pulmonary arteries. No calcium response was observed in cultured mouse dorsal root ganglion neurons or in naïve Chinese hamster ovary (CHO) cells exposed to oxaliplatin or cisplatin. However, oxaliplatin, and with lower potency, cisplatin, evoked a glutathione‐sensitive calcium response in CHO cells expressing mouse TRPA1. One single administration of oxaliplatin produced mechanical and cold hyperalgesia in rats, an effect selectively abated by the TRPA1 antagonist HC‐030031. Oxaliplatin administration caused mechanical and cold allodynia in mice. Both responses were absent in TRPA1‐deficient mice. Administration of cisplatin evoked mechanical allodynia, an effect that was reduced in TRPA1‐deficient mice. TRPA1 is therefore required for oxaliplatin‐evoked mechanical and cold hypersensitivity, and contributes to cisplatin‐evoked mechanical allodynia. Channel activation is most likely caused by glutathione‐sensitive molecules, including reactive oxygen species and their byproducts, which are generated after tissue exposure to platinum‐based drugs from cells surrounding nociceptive nerve terminals. TRPA1 is necessary and sufficient for mechanical‐ and cold‐hypersensitivity evoked by oxaliplatin/cisplatin. TRPA1 activation occurs through reactive molecules, after tissue exposure to platinum‐based drugs.

The Capsaicin Receptor TRPV1 Is a Crucial Mediator of the Noxious Effects of Mustard Oil

Mustard oil (MO) is a plant-derived irritant that has been extensively used in experimental models to induce pain and inflammation. The noxious effects of MO are currently ascribed to specific activation of the cation channel TRPA1 in nociceptive neurons. In contrast to this view, we show here that the capsaicin receptor TRPV1 has a surprisingly large contribution to aversive and pain responses and visceral irritation induced by MO. Furthermore, we found that this can be explained by previously unknown properties of this compound. First, MO has a bimodal effect on TRPA1, producing current inhibition at millimolar concentrations. Second, it directly and stably activates mouse and human recombinant TRPV1, as well as TRPV1 channels in mouse sensory neurons. Finally, physiological temperatures enhance MO-induced TRPV1 stimulation. Our results refute the dogma that TRPA1 is the sole nocisensor for MO and motivate a revision of the putative roles of these channels in models of MO-induced pain and inflammation. We propose that TRPV1 has a generalized role in the detection of irritant botanical defensive traits and in the coevolution of multiple mammalian and plant species.

GSK-3α/β kinases and amyloid production in vivo.

Arising from C. J. Phiel, C. A. Wilson, V. M.-Y. Lee & P. S. Klein 423, 435–439 (2003)10.1038/nature01640A major unresolved issue in Alzheimer’s disease is identifying the mechanisms that regulate proteolytic processing of amyloid precursor protein (APP)—glycogen synthase kinase-3 (GSK-3) isozymes are thought to be important in this regulation. Phiel et al. proposed that GSK-3α, but not GSK-3β, controls production of amyloid. We analysed the proteolytic processing of mouse and human APP in mouse brain in vivo in five different genetic and viral models. Our data do not yield evidence for either GSK-3α-mediated or GSK-3β-mediated control of APP processing in brain in vivo.

Mechanisms of TRPV1 Activation and Sensitization by Allyl Isothiocyanate

Allyl isothiocyanate (AITC; aka, mustard oil) is a powerful irritant produced by Brassica plants as a defensive trait against herbivores and confers pungency to mustard and wasabi. AITC is widely used experimentally as an inducer of acute pain and neurogenic inflammation, which are largely mediated by the activation of nociceptive cation channels transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1 (TRPV1). Although it is generally accepted that electrophilic agents activate these channels through covalent modification of cytosolic cysteine residues, the mechanism underlying TRPV1 activation by AITC remains unknown. Here we show that, surprisingly, AITC-induced activation of TRPV1 does not require interaction with cysteine residues, but is largely dependent on S513, a residue that is involved in capsaicin binding. Furthermore, AITC acts in a membrane-delimited manner and induces a shift of the voltage dependence of activation toward negative voltages, which is reminiscent of capsaicin effects. These data indicate that AITC acts through reversible interactions with the capsaicin binding site. In addition, we show that TRPV1 is a locus for cross-sensitization between AITC and acidosis in nociceptive neurons. Furthermore, we show that residue F660, which is known to determine the stimulation by low pH in human TRPV1, is also essential for the cross-sensitization of the effects of AITC and low pH. Taken together, these findings demonstrate that not all reactive electrophiles stimulate TRPV1 via cysteine modification and help understanding the molecular bases underlying the surprisingly large role of this channel as mediator of the algesic properties of AITC.

Allyl isothiocyanate sensitizes TRPV1 to heat stimulation

The powerful plant-derived irritant allyl isothiocyanate (AITC, aka mustard oil) induces hyperalgesia to heat in rodents and humans through mechanisms that are not yet fully understood. It is generally believed that AITC activates the broadly tuned chemosensory cation channel transient receptor potential cation channel subfamily A member 1 (TRPA1), triggering an inflammatory response that sensitizes the heat sensor transient receptor potential cation channel subfamily V member 1 (TRPV1). In the view of recent data demonstrating that AITC can directly activate TRPV1, we here explored the possibility that this compound sensitizes TRPV1 to heat stimulation in a TRPA1-independent manner. Patch-clamp recordings and intracellular Ca2+ imaging experiments in HEK293T cells over-expressing mouse TRPV1 revealed that the increase in channel activation induced by heating is larger in the presence of AITC than in control conditions. The analysis of the effects of AITC and heat on the current–voltage relationship of TRPV1 indicates that the mechanism of sensitization is based on additive shifts of the voltage dependence of activation towards negative voltages. Finally, intracellular Ca2+ imaging experiments in mouse sensory neurons isolated from Trpa1 KO mice yielded that AITC enhances the response to heat, specifically in the subpopulation expressing TRPV1. Furthermore, this effect was strongly reduced by the TRPV1 inhibitor capsazepine and virtually absent in neurons isolated from double Trpa1/Trpv1 KO mice. Taken together, these findings demonstrate that TRPV1 is a locus for cross sensitization between AITC and heat in sensory neurons and may help explaining, at least in part, the role of this channel in AITC-induced hyperalgesia to heat.

Modulation of the cold-activated cation channel TRPM8 by surface charge screening

TRPM8, a cation channel activated by cold and by cooling agents such as menthol and icilin, is critically involved in somatosensory cold sensation. Ion fluxes through TRPM8 are highly sensitive to changes in extracellular Ca2+ and pH, but the mechanisms underlying this type of modulation are poorly understood. Here we provide evidence that inhibition of TRPM8 currents by extracellular divalent cations and protons is due to surface charge screening. We demonstrate that increasing concentrations of divalent cations or protons cause parallel shifts of the voltage dependence of TRPM8 activation towards positive potentials. These shifts were interpreted using the Gouy–Chapman–Stern theory, yielding an estimate for the density of fixed negative surface charge between 0.0098 and 0.0126 equivalent charges per Å2. These results represent the first description of the effects of surface charge screening on a TRP channel and provide a straightforward explanation for the known effects of extracellular Ca2+ on cold‐sensitive neurons.

Differential Effects of Bitter Compounds on the Taste Transduction Channels TRPM5 and IP3 Receptor Type 3

Transient receptor potential cation channel subfamily M member 5 (TRPM5) is a Ca(2+)-activated nonselective cation channel involved in the transduction of sweet, bitter, and umami tastes. We previously showed that TRPM5 is a locus for the modulation of taste perception by temperature changes, and by quinine and quinidine, 2 bitter compounds that suppress gustatory responses. Here, we determined whether other bitter compounds known to modulate taste perception also affect TRPM5. We found that nicotine inhibits TRPM5 currents with an effective inhibitory concentration of ~1.3mM at -50 mV. This effect may contribute to the inhibitory effect of nicotine on gustatory responses in therapeutic and experimental settings, where nicotine is often employed at millimolar concentrations. In addition, it implies the existence of a TRPM5-independent pathway for the detection of nicotine bitterness. Nicotine seems to act from the extracellular side of the channel, reducing the maximal whole-cell conductance and inducing an acceleration of channel closure that leads to a negative shift of the activation curve. TRPM5 currents were unaffected by nicotines metabolite cotinine, the intensive sweetener saccharin or by the bitter xanthines caffeine, theobromine, and theophylline. We also tested the effects of bitter compounds on another essential element of the sweet taste transduction pathway, the type 3 IP3 receptor (IP3R3). We found that IP3R3-mediated Ca(2+) flux is slightly enhanced by nicotine, not affected by saccharin, modestly inhibited by caffeine, theobromine, and theophylline, and strongly inhibited by quinine. Our results demonstrate that bitter compounds have differential effects on key elements of the sweet taste transduction pathway, suggesting for heterogeneous mechanisms of bitter-sweet taste interactions.

Definition of two agonist types at the mammalian cold-activated channel TRPM8

Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. DOI: http://dx.doi.org/10.7554/eLife.17240.001