TRPV3 is a calcium-permeable temperature-sensitive cation channel

Transient receptor potential (TRP) proteins are cation-selective channels that function in processes as diverse as sensation and vasoregulation. Mammalian TRP channels that are gated by heat and capsaicin (>43 degrees C; TRPV1 (ref. 1)), noxious heat (>52 degrees C; TRPV2 (ref. 2)), and cooling (< 22 degrees C; TRPM8 (refs 3, 4)) have been cloned; however, little is known about the molecular determinants of temperature sensing in the range between approximately 22 degrees C and 40 degrees C. Here we have identified a member of the vanilloid channel family, human TRPV3 (hTRPV3) that is expressed in skin, tongue, dorsal root ganglion, trigeminal ganglion, spinal cord and brain. Increasing temperature from 22 degrees C to 40 degrees C in mammalian cells transfected with hTRPV3 elevated intracellular calcium by activating a nonselective cationic conductance. As in published recordings from sensory neurons, the current was steeply dependent on temperature, sensitized with repeated heating, and displayed a marked hysteresis on heating and cooling. On the basis of these properties, we propose that hTRPV3 is thermosensitive in the physiological range of temperatures between TRPM8 and TRPV1.     

Spider toxins activate the capsaicin receptor to produce inflammatory pain

Bites and stings from venomous creatures can produce pain and inflammation as part of their defensive strategy to ward off predators or competitors. Molecules accounting for lethal effects of venoms have been extensively characterized, but less is known about the mechanisms by which they produce pain. Venoms from spiders, snakes, cone snails or scorpions contain a pharmacopoeia of peptide toxins that block receptor or channel activation as a means of producing shock, paralysis or death. We examined whether these venoms also contain toxins that activate (rather than inhibit) excitatory channels on somatosensory neurons to produce a noxious sensation in mammals. Here we show that venom from a tarantula that is native to the West Indies contains three inhibitor cysteine knot (ICK) peptides that target the capsaicin receptor (TRPV1), an excitatory channel expressed by sensory neurons of the pain pathway. In contrast with the predominant role of ICK toxins as channel inhibitors, these previously unknown 'vanillotoxins' function as TRPV1 agonists, providing new tools for understanding mechanisms of TRP channel gating. Some vanillotoxins also inhibit voltage-gated potassium channels, supporting potential similarities between TRP and voltage-gated channel structures. TRP channels can now be included among the targets of peptide toxins, showing that animals, like plants (for example, chilli peppers), avert predators by activating TRP channels on sensory nerve fibres to elicit pain and inflammation.     

Novel mechanism of voltage-dependent gating in L-type calcium channels

Activation of voltage-dependent calcium channels by membrane depolarization triggers a variety of key cellular responses, such as contraction in heart and smooth muscle and exocytotic secretion in endocrine and nerve cells. Modulation of calcium channel gating is believed to be the mechanism by which several neurotransmitters, hormones and therapeutic agents mediate their effects on cell function. Here we describe a novel type of voltage-dependent equilibrium between different gating patterns of dihydropyridine-sensitive (L-type) cardiac Ca2+ channels. Strong depolarizations drive the channel from its normal gating pattern into a mode of gating characterized by long openings and high open probability. The rate constants for conversions between gating modes, estimated from single channel recordings, are much slower than normal channel opening and closing rates, but the equilibrium between modes is almost as steeply voltage-dependent as channel activation and deactivation at more negative potentials. This new mechanism of voltage-dependent gating can explain previous reports of activity-dependent Ca2+ channel potentiation in cardiac and other cells and forms a potent mechanism by which Ca2+ uptake into cells could be regulated.     

The menthol receptor TRPM8 is the principal detector of environmental cold

Sensory nerve fibres can detect changes in temperature over a remarkably wide range, a process that has been proposed to involve direct activation of thermosensitive excitatory transient receptor potential (TRP) ion channels. One such channel--TRP melastatin 8 (TRPM8) or cold and menthol receptor 1 (CMR1)--is activated by chemical cooling agents (such as menthol) or when ambient temperatures drop below approximately 26 degrees C, suggesting that it mediates the detection of cold thermal stimuli by primary afferent sensory neurons. However, some studies have questioned the contribution of TRPM8 to cold detection or proposed that other excitatory or inhibitory channels are more critical to this sensory modality in vivo. Here we show that cultured sensory neurons and intact sensory nerve fibres from TRPM8-deficient mice exhibit profoundly diminished responses to cold. These animals also show clear behavioural deficits in their ability to discriminate between cold and warm surfaces, or to respond to evaporative cooling. At the same time, TRPM8 mutant mice are not completely insensitive to cold as they avoid contact with surfaces below 10 degrees C, albeit with reduced efficiency. Thus, our findings demonstrate an essential and predominant role for TRPM8 in thermosensation over a wide range of cold temperatures, validating the hypothesis that TRP channels are the principal sensors of thermal stimuli in the peripheral nervous system.     

Heat activation of TRPM5 underlies thermal sensitivity of sweet taste

TRPM5, a cation channel of the TRP superfamily, is highly expressed in taste buds of the tongue, where it has a key role in the perception of sweet, umami and bitter tastes. Activation of TRPM5 occurs downstream of the activation of G-protein-coupled taste receptors and is proposed to generate a depolarizing potential in the taste receptor cells. Factors that modulate TRPM5 activity are therefore expected to influence taste. Here we show that TRPM5 is a highly temperature-sensitive, heat-activated channel: inward TRPM5 currents increase steeply at temperatures between 15 and 35 degrees C. TRPM4, a close homologue of TRPM5, shows similar temperature sensitivity. Heat activation is due to a temperature-dependent shift of the activation curve, in analogy to other thermosensitive TRP channels. Moreover, we show that increasing temperature between 15 and 35 degrees C markedly enhances the gustatory nerve response to sweet compounds in wild-type but not in Trpm5 knockout mice. The strong temperature sensitivity of TRPM5 may underlie known effects of temperature on perceived taste in humans, including enhanced sweetness perception at high temperatures and 'thermal taste', the phenomenon whereby heating or cooling of the tongue evoke sensations of taste in the absence of tastants.     

Anandamide and arachidonic acid use epoxyeicosatrienoic acids to activate TRPV4 channels

TRPV4 is a widely expressed cation channel of the 'transient receptor potential' (TRP) family that is related to the vanilloid receptor VR1 (TRPV1). It functions as a Ca2+ entry channel and displays remarkable gating promiscuity by responding to both physical stimuli (cell swelling, innoxious heat) and the synthetic ligand 4alphaPDD. An endogenous ligand for this channel has not yet been identified. Here we show that the endocannabinoid anandamide and its metabolite arachidonic acid activate TRPV4 in an indirect way involving the cytochrome P450 epoxygenase-dependent formation of epoxyeicosatrienoic acids. Application of 5',6'-epoxyeicosatrienoic acid at submicromolar concentrations activates TRPV4 in a membrane-delimited manner and causes Ca2+ influx through TRPV4-like channels in vascular endothelial cells. Activation of TRPV4 in vascular endothelial cells might therefore contribute to the relaxant effects of endocannabinoids and their P450 epoxygenase-dependent metabolites on vascular tone.     

Single sodium channels from rat brain incorporated into planar lipid bilayer membranes

A voltage- and time-dependent conductance for sodium ions is responsible for the generation of impulses in most nerve and muscle cells. Changes in the sodium conductance are produced by the opening and closing of many discrete transmembrane channels. We present here the first report of electrical recordings from voltage-dependent sodium channels incorporated into planar lipid bilayers. In bilayers with many channels, batrachotoxin (BTX) induced a steady-state sodium current that was blocked by saxitoxin (STX) at nanomolar concentrations. All channels appeared in the bilayer with their STX blocking sites facing the side of vesicle addition, allowing us to define that as the extracellular side. Current fluctuations due to the opening and closing of single BTX-activated sodium channels were voltage-dependent (unit conductance, 30 pS in 0.5 M NaCl): the channels closed at large hyperpolarizing potentials. Slower fluctuations of the same amplitude, due to the blocking and unblocking of individual channels, were seen after addition of STX. Block of the sodium channels by STX was voltage-dependent, with hyperpolarizing potentials favouring block. The voltage-dependent gating, ionic selectivity and neurotoxin sensitivity suggest that these are the channels that normally underlie the sodium conductance change during the nerve impulse.     

Polymer inaccessible volume changes during opening and closing of a voltage-dependent ionic channel

Osmotic stress can be used to estimate the internal volume change during the opening and closing of a voltage gated ionic channel. Mitochondrial voltage-dependent anion channels, from rat liver and from Neurospora, reconstituted into planar lipid bilayers show a change of 2 to 4 X 10(4) A3 in internal volume, a large change inconsistent with a blocking or local gating model but supporting models with major closure of the channel space.     

TRPV3 is a temperature-sensitive vanilloid receptor-like protein

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments--such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons--suggest the existence of thermosensitive receptors distinct from VR1. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39 degrees C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels.     

破解人类感知之谜

 戴维·朱利叶斯和阿尔登·帕塔普蒂安因发现TRPV1、TRPM8离子蛋白通道和PIEZO基因，“揭开了人类温度和触觉感受器的‘神秘面纱’”，荣获2021年诺贝尔生理学或医学奖。通过回顾2位科学家的成长经历、科研历程及在人类感知领域的研究成果，探讨了“痛感”对人体的利弊，分析了新科学仪器的应用与基础科研的关系。     

Low intracellular pH and chemical agents slow inactivation gating in sodium channels of muscle

Excitation of nerve or muscle requires an orderly opening and closing of molecular pores, the ionic channels, in the plasma membrane. During the action potential, Na channels are opened (activated) by the advancing wave of depolarisation, contributing a pulse of inward sodium current, and then are closed again (inactivated) by the continued depolarisation. As one approach both to obtaining molecular information on the Na channel and towards further defining the recently discovered kinetic interactions of the inactivation and activation gating steps, we have surveyed here the effects of chemical agents reported to slow or prevent Na channel inactivation. We find that many of the agents studied by others on invertebrate giant axons or vertebrate nerve act on our frog skeletal muscle preparation. In addition, we have discovered that simply lowering the intracellular pH nearly eliminates inactivation. The activation mechanism seems to resist modification.     

Requirement of TRPC channels in netrin-1-induced chemotropic turning of nerve growth cones

Ion channels formed by the TRP (transient receptor potential) superfamily of proteins act as sensors for temperature, osmolarity, mechanical stress and taste. The growth cones of developing axons are responsible for sensing extracellular guidance factors, many of which trigger Ca2+ influx at the growth cone; however, the identity of the ion channels involved remains to be clarified. Here, we report that TRP-like channel activity exists in the growth cones of cultured Xenopus neurons and can be modulated by exposure to netrin-1 and brain-derived neurotrophic factor, two chemoattractants for axon guidance. Whole-cell recording from growth cones showed that netrin-1 induced a membrane depolarization, part of which remained after all major voltage-dependent channels were blocked. Furthermore, the membrane depolarization was sensitive to blockers of TRP channels. Pharmacological blockade of putative TRP currents or downregulation of Xenopus TRP-1 (xTRPC1) expression with a specific morpholino oligonucleotide abolished the growth-cone turning and Ca2+ elevation induced by a netrin-1 gradient. Thus, TRPC currents reflect early events in the growth cone's detection of some extracellular guidance signals, resulting in membrane depolarization and cytoplasmic Ca2+ elevation that mediates the turning of growth cones.     

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.     

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.     

CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains

ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.     

Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila

Discriminating among sensory stimuli is critical for animal survival. This discrimination is particularly essential when evaluating whether a stimulus is noxious or innocuous. From insects to humans, transient receptor potential (TRP) channels are key transducers of thermal, chemical and other sensory cues. Many TRPs are multimodal receptors that respond to diverse stimuli, but how animals distinguish sensory inputs activating the same TRP is largely unknown. Here we determine how stimuli activating Drosophila TRPA1 are discriminated. Although Drosophila TRPA1 responds to both noxious chemicals and innocuous warming, we find that TRPA1-expressing chemosensory neurons respond to chemicals but not warmth, a specificity conferred by a chemosensory-specific TRPA1 isoform with reduced thermosensitivity compared to the previously described isoform. At the molecular level, this reduction results from a unique region that robustly reduces the channel's thermosensitivity. Cell-type segregation of TRPA1 activity is critical: when the thermosensory isoform is expressed in chemosensors, flies respond to innocuous warming with regurgitation, a nocifensive response. TRPA1 isoform diversity is conserved in malaria mosquitoes, indicating that similar mechanisms may allow discrimination of host-derived warmth--an attractant--from chemical repellents. These findings indicate that reducing thermosensitivity can be critical for TRP channel functional diversification, facilitating their use in contexts in which thermal sensitivity can be maladaptive.     

Blocker protection in the pore of a voltage-gated K+ channel and its structural implications

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow 'inner pore' seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.     

Relating ligand binding to activation gating in CNGA2 channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory signal transduction in photoreceptors and olfactory cells. Structurally, CNG channels are heterotetramers composed of either two or three homologue subunits. Although it is well established that activation is a cooperative process of these subunits, it remains unknown whether the cooperativity is generated by the ligand binding, the gating, or both, and how the subunits interact. In this study, the action of homotetrameric olfactory-type CNGA2 channels was studied in inside-out membrane patches by simultaneously determining channel activation and ligand binding, using the fluorescent cGMP analogue 8-DY547-cGMP as the ligand. At concentrations of 8-DY547-cGMP < 1 microM, steady-state binding was larger than steady-state activation, whereas at higher concentrations it was smaller, generating a crossover of the steady-state relationships. Global analysis of these relationships together with multiple activation time courses following cGMP jumps showed that four ligands bind to the channels and that there is significant interaction between the binding sites. Among the binding steps, the second is most critical for channel opening: its association constant is three orders of magnitude smaller than the others and it triggers a switch from a mostly closed to a maximally open state. These results contribute to unravelling the role of the subunits in the cooperative mechanism of CNGA2 channel activation and could be of general relevance for the action of other ion channels and receptors.     

A TRPV family ion channel required for hearing in Drosophila

The many types of insect ear share a common sensory element, the chordotonal organ, in which sound-induced antennal or tympanal vibrations are transmitted to ciliated sensory neurons and transduced to receptor potentials. However, the molecular identity of the transducing ion channels in chordotonal neurons, or in any auditory system, is still unknown. Drosophila that are mutant for NOMPC, a transient receptor potential (TRP) superfamily ion channel, lack receptor potentials and currents in tactile bristles but retain most of the antennal sound-evoked response, suggesting that a different channel is the primary transducer in chordotonal organs. Here we describe the Drosophila Nanchung (Nan) protein, an ion channel subunit similar to vanilloid-receptor-related (TRPV) channels of the TRP superfamily. Nan mediates hypo-osmotically activated calcium influx and cation currents in cultured cells. It is expressed in vivo exclusively in chordotonal neurons and is localized to their sensory cilia. Antennal sound-evoked potentials are completely absent in mutants lacking Nan, showing that it is an essential component of the chordotonal mechanotransducer.