Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a ‘grab and stand’ mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.

To adapt to habitat temperature, vertebrates have developed sophisticated physiological and ecological mechanisms through evolution. Transient receptor potential melastatin 8 (TRPM8) serves as the primary sensor for cold. However, how cold activates TRPM8 and how this sensor is tuned for thermal adaptation remain largely unknown. Here we established a molecular framework of how cold is sensed in TRPM8 with a combination of patch-clamp recording, unnatural amino acid imaging, and structural modeling. We first observed that the maximum cold activation of TRPM8 in eight different vertebrates (i.e., African elephant and emperor penguin) with distinct side-chain hydrophobicity (SCH) in the pore domain (PD) is tuned to match their habitat temperature. We further showed that altering SCH for residues in the PD with solventaccessibility changes leads to specific tuning of the cold response in TRPM8. We also observed that knockin mice expressing the penguin's TRPM8 exhibited remarkable tolerance to cold. Together, our findings suggest a paradigm of thermal adaptation in vertebrates, where the evolutionary tuning of the cold activation in the TRPM8 ion channel through altering SCH and solvent accessibility in its PD largely contributes to the setting of the cold-sensitive/ tolerant phenotype.TRPM8 | cold activation | pore domain | side-chain hydrophobicity | thermal adaptation T o survive and thrive, all living beings have to perceive and adapt to ambient temperature (1), which varies over a wide range from below −50°C in polar areas to above 50°C in deserts (2). Therefore, sophisticated physiological and ecological mechanisms have been developed through evolution to first detect and then adapt to ambient temperature (3-5). The transient receptor potential melastatin 8 (TRPM8) channel is the prototypical sensor for cold in vertebrates (6, 7), which has been validated in both knockout mice (8) and pharmacological studies (9). However, how cold activates TRPM8 remains obscure. From the perspective of channel structure, an earlier study suggested that its C terminus is crucial for cold activation (10), while subsequent work demonstrated that the transmembrane core domain (5) or the pore domain (11) is essential for setting cold response. Although high-resolution structures of TRPM8 have been resolved by cryoelectron microscopy in both the apo and ligand-bound states (12-14), its coldactivated state structure is still unavailable. From the perspective of thermodynamics, large enthalpic (ΔH) and entropic (ΔS) changes are associated with TRPM8 cold activation (15,16). Changes in heat capacity have also been hypothesized to mediate cold activation (17), though experimental evidence for such a hypothesis is limited to voltage-gated potassium channels (18). Interestingly, cold activation of TRPM8 is tuned during evolution in several tested vertebrate species (5,11,19). To understand both the structural and thermodynamic bases of TRPM8 cold activation, we attempted to gain insights from TRPM8 orthologs in vertebrate species inhabiti...

SignificanceThe work showed that centipede venom can cause disorders in cardiovascular, respiratory, and nervous systems. The cardiovascular toxicity of the venom comes mostly from a peptide toxin SsTx, which blocks the KCNQ family of potassium channels. Retigabine, a KCNQ channel opener, neutralizes centipede venom toxicity, and thus could be used to treat centipede envenomation.

Transient receptor potential vanilloid 6 (TRPV6), a calcium-selective channel possessing six transmembrane domains (S1-S6) and intracellular N-/C-termini, plays crucial roles in calcium absorption in epithelia and bone and is involved in human diseases including vitamin-D deficiency, osteoporosis and cancer. The TRPV6 function and regulation remain poorly understood. Here by electrophysiology and Xenopus oocyte expression we found that Arg510 in the S4-S5 linker and Trp633 in the TRP helix are functionally critical. By coimmunoprecipitation and in vitro binding we found that the Arg510:Trp633 pair mediates binding between the linker (L) and C-terminal TRP helix (C) and that disruption of the linker/TRP helix (L/C) interaction substantially increases the channel activity. We also found that Trp361 in the N-terminal pre-S1 helix (N) and Ile637 mediate the pre-S1/TRP helix (N/C) binding which is functionally critical as well. Calcium imaging in human embryonic kidney cells further supported functional importance of Trp361 and Trp633. Besides, disruption of either interaction by blocking peptides activated TRPV6. The L/C interaction was required for the N/C interaction but not reversely. Together, we showed that the TRPV6 intramolecular S4-S5 linker to TRP helix and pre-S1 helix to TRP helix interactions, mediated by Arg510:Trp633 and Trp361:Ile637 bonding, respectively, are autoinhibitory and are required for maintaining TRPV6 at basal states. This study reveals a regulatory mechanism of TRPV6 activation-autoinhibition, which would help elucidating the corresponding mechanisms in other TRP channels. Supported by AIGSS (to RC), NSERC and KFoC (to XZC).

Significance Adaptation to more severe ambient temperature fluctuations can be considered one of the key innovations of terrestrial tetrapods. Our study shows the formation of the functional MHR1-3 domain in transient receptor potential melastatin 8 (TRPM8) bestowed the channel with cold sensitivity during the water-to-land transition. The evolved MHR1-3 domain found in terrestrial tetrapods serves as an independent apparatus with cold sensitivity. Furthermore, this domain with independent cold sensitivity is necessary for the regulatory mechanism of the pore domain, where the efficacy of cold activation is largely altered by evolutionary tuning of the hydrophobicity of several residues during the diversification of terrestrial tetrapods. Our findings advance the understanding of cold-sensing emergence during evolution and the thermodynamic basis of TRPM8 cold activation.

We report on local magnetization, tunnel diode oscillators, and specific-heat measurements in a series of Ba(Ni x Fe 1−x ) 2 As 2 single crystals (0.26 x 0.74). We show that the London penetration depth λ(T ) = λ(0) + λ(T ) scales as λ (0) in both underdoped and overdoped samples. Moreover, the slope of the upper critical field [H c2 = −(dH c2 /dT ) |T →Tc ] decreases with T c in overdoped samples but increases with decreasing T c in underdoped samples. The remarkable variation of λ(0) with T c and the nonexponential temperature dependence of λ clearly indicates that pair-breaking effects are important in this system. We show that the observed scalings strongly suggest that those pair-breaking effects could be associated with quantum fluctuations near three-dimensional superconducting critical points.

Transient receptor potential vanilloid 1 (TRPV1) ion channel is a nociceptor critically involved in pain sensation. Direct blockade of TRPV1 exhibits significant analgesic effects but also incurs severe side effects such as hyperthermia, causing failures of TRPV1 inhibitors in clinical trials. In order to selectively target TRPV1 channels that are actively involved in pain-sensing, peptidic positive allosteric modulators (PAMs) based on the high-resolution structure of the TRPV1 intracellular ankyrin-repeat like domain are de novo designed. The hotspot centric approach is optimized for protein design; its usage in Rosetta increases the success rate in protein binder design. It is demonstrated experimentally, with a combination of fluorescence resonance energy transfer (FRET) imaging, surface plasmon resonance, and patch-clamp recording, that the designed PAMs bind to TRPV1 with nanomolar affinity and allosterically enhance its response to ligand activation as it is designed. It is further demonstrated that the designed PAM exhibits long-lasting in vivo analgesic effects in rats without changing their body temperature, suggesting that they have potentials for developing into novel analgesics.

Animal toxins that are used to subdue prey and deter predators act as the key drivers in natural food chains and ecosystems. However, the predators of venomous animals may exploit feeding adaptation strategies to overcome toxins their prey produce. Much remains unknown about the genetic and molecular game process in the toxin-dominant food chain model. Here, we show an evolutionary strategy in different trophic levels of scorpion-eating amphibians, scorpions and insects, representing each predation relationship in habitats dominated by the paralytic toxins of scorpions. For scorpions preying on insects, we found that the scorpion α-toxins irreversibly activate the skeletal muscle sodium channel of their prey (insect, BgNaV1) through a membrane delivery mechanism and an efficient binding with the Asp/Lys-Tyr motif of BgNaV1. However, in the predatory game between frogs and scorpions, with a single point mutation (Lys to Glu) in this motif of the frog's skeletal muscle sodium channel (fNaV1.4), fNaV1.4 breaks this interaction and diminishes muscular toxicity to the frog; thus, frogs can regularly prey on scorpions without showing paralysis. Interestingly, this molecular strategy also has been employed by some other scorpion-eating amphibians, especially anurans. In contrast to these amphibians, the Asp/Lys-Tyr motifs are structurally and functionally conserved in other animals that do not prey on scorpions. Together, our findings elucidate the protein-protein interacting mechanism of a toxin-dominant predator-prey system, implying the evolutionary game theory at a molecular level.