Synthetic cation channel: reconstructing the ion permeation pathway of TRPA1 in an artificial system† † Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra05676k
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ABSTRACT:
SUBMITTER: Xin P
PROVIDER: S-EPMC11348841 | biostudies-literature | 2024 Aug
REPOSITORIES: biostudies-literature
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Project description:The Ca(2+)-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca(2+) ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of approximately 11 A. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca(2+), and at a membrane potential of -80 mV, approximately 17% of the inward TRPA1 current is carried by Ca(2+). Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 A and an increase in divalent cation selectivity and fractional Ca(2+) current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca(2+) current also prevented mustard-oil-induced increases in Ca(2+) permeation. It is interesting that fractional Ca(2+) currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca(2+) in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca(2+) influx.
| S-EPMC2830466 | biostudies-literature
Project description:RationaleTransient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.ObjectiveTo identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.Methods and resultsFreshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca2+ concentration ([Ca2+]i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca2+]i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca2+]i and velocity of shortening as well as an acceleration in [Ca2+]i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1-/- mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14-22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) (1-5 min) that correlated with increases in CM [Ca2+]i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca2+]i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L).ConclusionsThese novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca2+ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.
| S-EPMC5786180 | biostudies-literature
Project description:Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.
| S-EPMC7926781 | biostudies-literature
Project description:While most animals thermotax only to regulate their temperature, female mosquitoes are attracted to human body heat during pursuit of a blood meal. Here we elucidate the basic rules of Aedes aegypti thermotaxis and test the function of candidate thermoreceptors in this important behavior. We show that host-seeking mosquitoes are maximally attracted to thermal stimuli approximating host body temperatures, seeking relative warmth while avoiding both relative cool and stimuli exceeding host body temperature. We found that the cation channel TRPA1, in addition to playing a conserved role in thermoregulation and chemosensation, is required for this specialized host-selective thermotaxis in mosquitoes. During host-seeking, AaegTRPA1(-/-) mutants failed to avoid stimuli exceeding host temperature, and were unable to discriminate between host-temperature and high-temperature stimuli. TRPA1-dependent tuning of thermotaxis is likely critical for mosquitoes host-seeking in a complex thermal environment in which humans are warmer than ambient air, but cooler than surrounding sun-warmed surfaces.
| S-EPMC4718722 | biostudies-literature
Project description: Not available
| EMPIAR-10024 | biostudies-other
Project description:Calcium is the most abundant metal in the human body that plays vital roles as a cellular electrolyte as well as the smallest and most frequently used signaling molecule. Calcium uptake in epithelial tissues is mediated by tetrameric calcium-selective transient receptor potential (TRP) channels TRPV6 that are implicated in a variety of human diseases, including numerous forms of cancer. We used TRPV6 crystal structures as templates for molecular dynamics simulations to identify ion binding sites and to study the permeation mechanism of calcium and other ions through TRPV6 channels. We found that at low Ca2+ concentrations, a single calcium ion binds at the selectivity filter narrow constriction formed by aspartates D541 and allows Na+ permeation. In the presence of ions, no water binds to or crosses the pore constriction. At high Ca2+ concentrations, calcium permeates the pore according to the knock-off mechanism that includes formation of a short-lived transition state with three calcium ions bound near D541. For Ba2+, the transition state lives longer and the knock-off permeation occurs slower. Gd3+ binds at D541 tightly, blocks the channel and prevents Na+ from permeating the pore. Our results provide structural foundations for understanding permeation and block in tetrameric calcium-selective ion channels.
| S-EPMC5890290 | biostudies-literature
Project description:The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modulated by myriad noxious stimuli that interact with multiple regions of the channel, including cysteine-reactive natural extracts from onion and garlic which modify residues in the cytoplasmic domains. The way in which TRPA1 cytoplasmic domain modification is coupled to opening of the ion-conducting pore has yet to be elucidated. The cryo-EM structure of TRPA1 revealed a tetrameric C-terminal coiled-coil surrounded by N-terminal ankyrin repeat domains (ARDs), an architecture shared with the canonical transient receptor potential (TRPC) ion channel family. Similarly, structures of the TRP melastatin (TRPM) ion channel family also showed a C-terminal coiled-coil surrounded by N-terminal cytoplasmic domains. This conserved architecture may indicate a common gating mechanism by which modification of cytoplasmic domains can transduce conformational changes to open the ion-conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal coiled-coil domains can be expressed in bacteria and maintain the ability to interact. We tested three gating regulators: temperature; the polyphosphate compound IP6; and the covalent modifier allyl isothiocyanate to determine whether they alter N- and C-terminal interactions. We found that none of the modifiers tested abolished ARD-coiled-coil interactions, though there was a significant reduction at 37˚C. We found that coiled-coils tetramerize in a concentration dependent manner, with monomers and trimers observed at lower concentrations. Our system provides a method for examining the mechanism of oligomerization of TRPA1 cytoplasmic domains as well as a system to study the transmission of conformational changes resulting from covalent modification.
| S-EPMC6386368 | biostudies-literature
Project description:The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to ∼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents.
| S-EPMC4409540 | biostudies-literature
Project description:Ion channels catalyze ionic permeation across membranes via water-filled pores. To understand how changes in intracellular magnesium concentration regulate the influx of Mg2+ into cells, we examine early events in the relaxation of Mg2+ channel CorA toward its open state using massively-repeated molecular dynamics simulations conducted either with or without regulatory ions. The pore of CorA contains a 2-nm-long hydrophobic bottleneck which remained dehydrated in most simulations. However, rapid hydration or "wetting" events concurrent with small-amplitude fluctuations in pore diameter occurred spontaneously and reversibly. In the absence of regulatory ions, wetting transitions are more likely and include a wet state that is significantly more stable and more hydrated. The free energy profile for Mg2+ permeation presents a barrier whose magnitude is anticorrelated to pore diameter and the extent of hydrophobic hydration. These findings support an allosteric mechanism whereby wetting of a hydrophobic gate couples changes in intracellular magnesium concentration to the onset of ionic conduction.
| S-EPMC4504495 | biostudies-literature
Project description:KEY POINTS:Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na+ , K+ and Ca2+ channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K+ channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway. ABSTRACT:Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K+ channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.
| S-EPMC6002228 | biostudies-literature