Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12′ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12′ lysines in the α, γ and δ subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in β or ε subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12′ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of α subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channe

Structure and function of the nicotinic acetylcholine receptor

The subject of this thesis is the study of the structure and function of the human muscle nicotinic acetylcholine receptor (nAChR), which is a typical member of the large class of ligand-gated ion channels. Appropriate techniques, like site-directed mutagenesis, patch-clamp and fluorescence microscopy, and combinations thereof, were applied to gain insight into selected aspects of the structure-function relationship of this receptor. Special emphasis was put on the investigation of single receptors, wherever this promised scientific benefit. Experiments with the aim to study structural rearrangements of nAChR upon ligand binding by fluorescence resonance energy transfer (FRET) were undertaken. Such intramolecular motions are expected to link ligand binding to channel gating and are therefore fundamental to understand the function of nAChR. Double-cysteine mutants were produced and receptors were labelled on living cells in order to measure changes in FRET efficiency due to ligand binding. A novel class of functionally modified ligands for the nAChR was characterized by patch-clamp technique. Derivatives that carry a thiol-reactive group were shown to be full and very potent agonists. On the bases of a structural model of the binding site, residues were selected, that are likely to be in contact with the bound ligand. Equivalent single-cysteine mutants were produced to covalently bind these agonists and thereby to gain information about the mechanisms of ligand binding and receptor activation. Ligands that were coupled to fluorophores turned out to be either partial or full agonists with nanomolar efficacies for the nAChR. Their use to study the diffusion of single nAChR on living HEK293-cells for different functional states is described in detail. The dependency of the lateral diffusion on the activation state (closed/opened/desensitized) was studied. Additionally these ligands served to demonstrate the feasibility to simultaneously combine patch-clamp and fluorescent binding experiments on a single-molecule level. Acetylcholine receptors carrying a single point-mutation, related to the congenital myasthenic syndrome, were found to stabilize an intermediate conductance state and were studied in detail by single-channel electrophysiology. The dependency of the gating behavior on different ligands, ligand concentration and transmembrane voltage was investigated. Mutants, carrying equivalent mutations on different subunits were produced to reveal the origin of this effect. Quantitative characterization was obtained by dwell-time and spectral noise-analysis, in order to gain insight on sub-millisecond fluctuations of the channel lining transmembrane regions of the nAChR and the gating mechanism in general

The pore-domain of TRPA1 mediates the inhibitory effect of the antagonist 6-methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole.

The transient receptor potential ion channel TRPA1 confers the ability to detect tissue damaging chemicals to sensory neurons and as a result mediates chemical nociception in vivo. Mouse TRPA1 is activated by electrophilic compounds such as mustard-oil and several physical stimuli such as cold temperature. Due to its sensory function inhibition of TRPA1 activity might provide an effective treatment against chronic and inflammatory pain. Therefore, TRPA1 has become a target for the development of analgesic drugs. 6-Methyl-5-(2-(trifluoromethyl)phenyl)-1H-indazole (Compound 31) has been identified by a chemical screen and lead optimization as an inhibitor of chemical activation of TRPA1. However, the structures or domains of TRPA1 that mediate the inhibitory effect of Compound 31 are unknown. Here, we screened 12,000 random mutant clones of mouse TRPA1 for their sensitivity to mustard-oil and the ability of Compound 31 to inhibit chemical activation by mustard-oil. In addition, we separately screened this mutant library while stimulating it with cold temperatures. We found that the single-point mutation I624N in the N-terminus of TRPA1 specifically affects the sensitivity to mustard-oil, but not to cold temperatures. This is evidence that sensitivity of TRPA1 to chemicals and cold temperatures is conveyed by separable mechanisms. We also identified five mutations located within the pore domain that cause loss of inhibition by Compound 31. This result demonstrates that the pore-domain is a regulator of chemical activation and suggests that Compound 31 might be acting directly on the pore-domain

Single Residues in the Outer Pore of TRPV1 and TRPV3 Have Temperature-Dependent Conformations

<div><p>Thermosensation is mediated by ion channels that are highly temperature-sensitive. Several members of the family of transient receptor potential (TRP) ion channels are activated by cold or hot temperatures and have been shown to function as temperature sensors in vivo. The molecular mechanism of temperature-sensitivity of these ion channels is not understood. A number of domains or even single amino acids that regulate temperature-sensitivity have been identified in several TRP channels. However, it is unclear what precise conformational changes occur upon temperature activation. Here, we used the cysteine accessibility method to probe temperature-dependent conformations of single amino acids in TRP channels. We screened over 50 amino acids in the predicted outer pore domains of the heat-activated ion channels TRPV1 and TRPV3. In both ion channels we found residues that have temperature-dependent accessibilities to the extracellular solvent. The identified residues are located within the second predicted extracellular pore loop. These residues are identical or proximal to residues that were shown to be specifically required for temperature-activation, but not chemical activation. Our data precisely locate conformational changes upon temperature-activation within the outer pore domain. Collectively, this suggests that these specific residues and the second predicted pore loop in general are crucial for the temperature-activation mechanism of these heat-activated thermoTRPs.</p> </div

Homology model of the pore-domain of TRPA1.

<p>A) Amino-acid sequence of mouse TRPA1 pore-domain. Single-point mutations reducing inhibition by Compound 31 are highlighted in red and structural domains of the pore are indicated below. B) Structural model of the mouse TRPA1 pore-domain. Single-point mutations reducing inhibition by Compound 31 are highlighted in red.</p

Single-point mutations reduce inhibition by Compound 31.

<p>A) Concentration-response curves of wild-type TRPA1 and single-point mutants obtained from pre-incubation of Compound 31 and subsequent stimulation by 100 µM mustard-oil. All curves are normalized to maximal responses evoked by mustard-oil in the absence of Compound 31. Error bars are stdv., n = 6. Lines are fits of Hill-equations to the data. B) Concentration-response curves of wild-type TRPA1, pcDNA and single-point mutants to mustard-oil (final concentrations). Error bars are stdv., n = 4. Lines are fits of Hill-equations to the data.</p

Transduction of Repetitive Mechanical Stimuli by Piezo1 and Piezo2 Ion Channels

Summary: Several cell types experience repetitive mechanical stimuli, including vein endothelial cells during pulsating blood flow, inner ear hair cells upon sound exposure, and skin cells and their innervating dorsal root ganglion (DRG) neurons when sweeping across a textured surface or touching a vibrating object. While mechanosensitive Piezo ion channels have been clearly implicated in sensing static touch, their roles in transducing repetitive stimulations are less clear. Here, we perform electrophysiological recordings of heterologously expressed mouse Piezo1 and Piezo2 responding to repetitive mechanical stimulations. We find that both channels function as pronounced frequency filters whose transduction efficiencies vary with stimulus frequency, waveform, and duration. We then use numerical simulations and human disease-related point mutations to demonstrate that channel inactivation is the molecular mechanism underlying frequency filtering and further show that frequency filtering is conserved in rapidly adapting mouse DRG neurons. Our results give insight into the potential contributions of Piezos in transducing repetitive mechanical stimuli. : Lewis et al. examine how Piezo1 and Piezo2 mechanosensitive ion channels transduce repetitive mechanical stimulations. They find that Piezos act as pronounced frequency filters, via a mechanism requiring intact channel inactivation. Keywords: mechanotransduction, Piezo1, Piezo2, mechanosensitive ion channel, repetitive stimulation, frequency filtering, inactivation, four-state gating mechanism, dorsal root ganglia neuron

Temperature-dependent labeling in TRPV1.

<p>(A) Temperature as a function of time during FLIPR temperature-activation assay. (B–D) Representative examples of fluorescence responses upon temperature stimulation of TRPV1 N652C (B), A657C (C) and Y53C (D) after incubation of MTSET at 20°C (blue) and 42°C (red). For both temperatures, buffer as a negative control is colored gray; n>8 wells. Error bars are 2× s.e. (E) Average basal fluorescence change of TRPV1 Y653C after incubation of MTSET at 20°C and 42°C. The basal fluorescence is averaged fluorescence level between 0 to 20 sec. For each experiment, the increase of basal fluorescence was obtained by subtracting buffer control from MTSET incubation. Five independent experiments were performed for 20°C and three for 40°C with n>5 wells per experiment. Two-tailed t-test, *p = 0.036. Error bars are mean ± s.e.</p

Temperature-dependent labeling in TRPV3.

<p>(A) Temperature as a function of time during FLIPR temperature-activation assay. (B) Representative examples of fluorescence responses upon temperature stimulation of TRPV3 I652C. n>8 wells. Error bars are mean ±2x s.e. (C) Average basal fluorescence change of TRPV3 I652C after incubation of MTSET at 20°C and 40°C. For each experiment, the basal fluorescence is an average of fluorescence between 0 and 20 sec. Fluorescence change is the difference of MTSET incubation and buffer incubation basal fluorescence. Numbers of independent experiments are shown in the bar graph and n>8 wells per experiment. Error bars are mean ± s.e. Two-tailed t-test, ***p<0.0001. (D) Representative examples of fluorescence responses upon temperature stimulation of L655, n>8 wells, Error bars are mean ±2× s.e. (E) Average basal fluorescence change of TRPV3 L655C after incubation of MTSET at 20°C and 40°C. Number of experiments is shown in the bar graph and n>8 wells per experiment. Error bars are mean ± s.e. Two-tailed t-test, **p = 0.0062. (F) The basal fluorescence change of TRPV3 L655C after incubation of MTSET at 20°C and 40°C as a function of MTSET concentration. The incubation time was 10 min. n>5 wells. Error bars are mean ± s.d. Straight lines are exponential fits to the data.</p

Location of temperature-dependent accessible residues.

<p>(A) Sequence alignment of pore domains of rat TRPV1 and mouse TRPV3. Predicted structural domains are indicated above. Residues with temperature-independent accessibility are highlighted in cyan and residues with temperature-dependent accessibility are in pink. (B) Homology models of pore domains. Same color coding was used as the above.</p