The transient receptor potential (TRP) ion channel family is large and functionally diverse, second only to potassium channels. Despite their prominence within the animal kingdom, TRP channels have resisted crystallization and structural determination for many years. This barrier was recently broken when the three-dimensional structure of the vanilloid receptor 1 (TRPV1) was determined by single particle electron cryo-microscopy (cryo-EM). Moreover, this is the first example in which the near atomic resolution structure of an integral membrane protein was elucidated by this technique and in a manner not requiring crystals, demonstrating the transformative power of single particle cryo-EM for revealing high-resolution structures of integral membrane proteins, particularly those of mammalian origin. Here we summarize technical advances, in both biochemistry and cryo-EM, that led to this major breakthrough.
Acid-sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the superfamily of degenerin/epithelial sodium channels. Here, we determine the cocrystal structure of chicken ASIC1a with MitTx, a pain-inducing toxin from the Texas coral snake, to define the structure of the open state of ASIC1a. In the MitTx-bound open state and in the previously determined low-pH desensitized state, TM2 is a discontinuous ? helix in which the Gly-Ala-Ser selectivity filter adopts an extended, belt-like conformation, swapping the cytoplasmic one-third of TM2 with an adjacent subunit. Gly 443 residues of the selectivity filter provide a ring of three carbonyl oxygen atoms with a radius of ?3.6 Å, presenting an energetic barrier for hydrated ions. The ASIC1a-MitTx complex illuminates the mechanism of MitTx action, defines the structure of the selectivity filter of voltage-independent, sodium-selective ion channels, and captures the open state of an ASIC.
Nociception is the process whereby primary afferent nerve fibers of the somatosensory system detect noxious stimuli. Pungent irritants from pepper, mint, and mustard plants have served as powerful pharmacological tools for identifying molecules and mechanisms underlying this initial step of pain sensation. These natural products have revealed three members of the transient receptor potential (TRP) ion channel family--TRPV1, TRPM8, and TRPA1--as molecular detectors of thermal and chemical stimuli that activate sensory neurons to produce acute or persistent pain. Analysis of TRP channel function and expression has validated the existence of nociceptors as a specialized group of somatosensory neurons devoted to the detection of noxious stimuli. These studies are also providing insight into the coding logic of nociception and how specification of nociceptor subtypes underlies behavioral discrimination of noxious thermal, chemical, and mechanical stimuli. Biophysical and pharmacological characterization of these channels has provided the intellectual and technical foundation for developing new classes of analgesic drugs.
Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (consisting of transmembrane segments 1-4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels.
Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4?Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular mouth with a short selectivity filter. The conserved TRP domain interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.
The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels.
Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide array of physical and chemical stimuli, making them important components of sensory systems in both vertebrate and invertebrate organisms. Mammalian TRPA1 channels are activated by chemically reactive irritants, whereas snake and Drosophila TRPA1 orthologs are preferentially activated by heat. By comparing human and rattlesnake TRPA1 channels, we have identified two portable heat-sensitive modules within the ankyrin repeat-rich aminoterminal cytoplasmic domain of the snake ortholog. Chimeric channel studies further demonstrate that sensitivity to chemical stimuli and modulation by intracellular calcium also localize to the N-terminal ankyrin repeat-rich domain, identifying this region as an integrator of diverse physiological signals that regulate sensory neuron excitability. These findings provide a framework for understanding how restricted changes in TRPA1 sequence account for evolution of physiologically diverse channels, also identifying portable modules that specify thermosensitivity.
Natural products that elicit discomfort or pain represent invaluable tools for probing molecular mechanisms underlying pain sensation. Plant-derived irritants have predominated in this regard, but animal venoms have also evolved to avert predators by targeting neurons and receptors whose activation produces noxious sensations. As such, venoms provide a rich and varied source of small molecule and protein pharmacophores that can be exploited to characterize and manipulate key components of the pain-signalling pathway. With this in mind, here we perform an unbiased in vitro screen to identify snake venoms capable of activating somatosensory neurons. Venom from the Texas coral snake (Micrurus tener tener), whose bite produces intense and unremitting pain, excites a large cohort of sensory neurons. The purified active species (MitTx) consists of a heteromeric complex between Kunitz- and phospholipase-A2-like proteins that together function as a potent, persistent and selective agonist for acid-sensing ion channels (ASICs), showing equal or greater efficacy compared with acidic pH. MitTx is highly selective for the ASIC1 subtype at neutral pH; under more acidic conditions (pH < 6.5), MitTx massively potentiates (>100-fold) proton-evoked activation of ASIC2a channels. These observations raise the possibility that ASIC channels function as coincidence detectors for extracellular protons and other, as yet unidentified, endogenous factors. Purified MitTx elicits robust pain-related behaviour in mice by activation of ASIC1 channels on capsaicin-sensitive nerve fibres. These findings reveal a mechanism whereby snake venoms produce pain, and highlight an unexpected contribution of ASIC1 channels to nociception.
Primary afferent "pain" fibers (nociceptors) are divided into subclasses based on distinct molecular and anatomical features, and these classes mediate noxious modality-specific contributions to behaviors evoked by painful stimuli. Whether the heat and capsaicin receptor transient receptor potential vanilloid-1 (TRPV1) is expressed heterogeneously across several sensory populations, or is selectively expressed by a unique nociceptor subclass, however, is unclear. Here we used two lines of Trpv1 reporter mice to investigate the primary afferent expression of TRPV1, both during development and in the adult. We demonstrate, using Cre-induced lineage tracing, that during development TRPV1 is transiently expressed in a wide range of dorsal root ganglion neurons, and that its expression is gradually refined, such that TRPV1 transcripts become restricted to a specific subset of peptidergic sensory neurons. Finally, the remarkable sensitivity that is characteristic of these reporter mice revealed an innervation of central and peripheral targets by TRPV1+ primary afferents in the adult that is considerably more extensive than has previously been appreciated.
The objective of this article is to describe and show a number of imaging findings that are classical for an intussusception in an adult and discuss the clinical manifestations and associated findings. We also discuss the differential diagnosis of intussusception in an adult and the most likely causes.
The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.
Vampire bats (Desmodus rotundus) are obligate blood feeders that have evolved specialized systems to suit their sanguinary lifestyle. Chief among such adaptations is the ability to detect infrared radiation as a means of locating hotspots on warm-blooded prey. Among vertebrates, only vampire bats, boas, pythons and pit vipers are capable of detecting infrared radiation. In each case, infrared signals are detected by trigeminal nerve fibres that innervate specialized pit organs on the animals face. Thus, vampire bats and snakes have taken thermosensation to the extreme by developing specialized systems for detecting infrared radiation. As such, these creatures provide a window into the molecular and genetic mechanisms underlying evolutionary tuning of thermoreceptors in a species-specific or cell-type-specific manner. Previously, we have shown that snakes co-opt a non-heat-sensitive channel, vertebrate TRPA1 (transient receptor potential cation channel A1), to produce an infrared detector. Here we show that vampire bats tune a channel that is already heat-sensitive, TRPV1, by lowering its thermal activation threshold to about 30?°C. This is achieved through alternative splicing of TRPV1 transcripts to produce a channel with a truncated carboxy-terminal cytoplasmic domain. These splicing events occur exclusively in trigeminal ganglia, and not in dorsal root ganglia, thereby maintaining a role for TRPV1 as a detector of noxious heat in somatic afferents. This reflects a unique organization of the bat Trpv1 gene that we show to be characteristic of Laurasiatheria mammals (cows, dogs and moles), supporting a close phylogenetic relationship with bats. These findings reveal a novel molecular mechanism for physiological tuning of thermosensory nerve fibres.
Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a thermal image of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibres of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibres that innervate the pit organ. TRPA1 orthologues from pit-bearing snakes (vipers, pythons and boas) are the most heat-sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of transient receptor potential (TRP) channels as thermosensors in the vertebrate nervous system.
Toxins have evolved to target regions of membrane ion channels that underlie ligand binding, gating, or ion permeation, and have thus served as invaluable tools for probing channel structure and function. Here, we describe a peptide toxin from the Earth Tiger tarantula that selectively and irreversibly activates the capsaicin- and heat-sensitive channel, TRPV1. This high-avidity interaction derives from a unique tandem repeat structure of the toxin that endows it with an antibody-like bivalency. The "double-knot" toxin traps TRPV1 in the open state by interacting with residues in the presumptive pore-forming region of the channel, highlighting the importance of conformational changes in the outer pore region of TRP channels during activation.
The nervous system detects and interprets a wide range of thermal and mechanical stimuli, as well as environmental and endogenous chemical irritants. When intense, these stimuli generate acute pain, and in the setting of persistent injury, both peripheral and central nervous system components of the pain transmission pathway exhibit tremendous plasticity, enhancing pain signals and producing hypersensitivity. When plasticity facilitates protective reflexes, it can be beneficial, but when the changes persist, a chronic pain condition may result. Genetic, electrophysiological, and pharmacological studies are elucidating the molecular mechanisms that underlie detection, coding, and modulation of noxious stimuli that generate pain.
Animals sense changes in ambient temperature irrespective of whether core body temperature is internally maintained (homeotherms) or subject to environmental variation (poikilotherms). Here we show that a cold-sensitive ion channel, TRPM8, displays dramatically different thermal activation ranges in frogs versus mammals or birds, consistent with variations in these species cutaneous and core body temperatures. Thus, somatosensory receptors are not static through evolution, but show functional diversity reflecting the characteristics of an organisms ecological niche.
This paper describes the synthesis of ion-pair-reinforced semi-interpenetrating polymer networks (SIPNs) as proton exchange membranes (PEMs) for the direct methanol fuel cells (DMFCs). Specifically, sulfonated poly(2,6-dimethyl-1,4-phenylene oxide) (SPPO), a linear polymer proton source, was immobilized in a brominated PPO (BPPO) network covalently cross-linked by ethylenediamine (EDA). The immobilization of SPPO in the SIPN network was accomplished not only by the usual means of mechanical interlocking but also by ion pair formation between the sulfonic acid groups of SPPO and the amine moieties formed during the cross-linking reaction of BPPO with EDA. Through the ion pair interactions, the immobilization of SPPO polymer in the BPPO network was made more effective, resulting in a greater uniformity of sulfonic acid cluster distribution in the membrane. The hydrophilic amine-containing cross-links also compensated for some of the decrease in proton conductivity caused by ion pair formation. The SIPN membranes prepared as such showed good proton conductivity, low methanol permeability, good mechanical properties, and dimensional stability. Consequently, the PPO based SIPN membranes were able to deliver a higher maximum power density than Nafion, demonstrating the potential of the SIPN structure for PEM designs.
Venoms often target vital processes to cause paralysis or death, but many types of venom also elicit notoriously intense pain. While these pain-producing effects can result as a byproduct of generalized tissue trauma, there are now multiple examples of venom-derived toxins that target somatosensory nerve terminals in order to activate nociceptive (pain-sensing) neural pathways. Intriguingly, investigation of the venom components that are responsible for evoking pain has revealed novel roles and/or configurations of well-studied toxin motifs. This review serves to highlight pain-producing toxins that target the capsaicin receptor, TRPV1, or members of the acid-sensing ion channel family, and to discuss the utility of venom-derived multivalent and multimeric complexes.
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