scholarly journals Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

2017 ◽  
Author(s):  
Ximena Steinberg ◽  
Marina A. Kasimova ◽  
Deny Cabezas-Bratesco ◽  
Jason Galpin ◽  
Ernesto Ladrón-de-Guevara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ion Selectivity ◽  
Noise Analysis ◽  
Conformational Dynamics ◽  
Selectivity Filter ◽  
Optical Data ◽  
Transient Receptor Potential Vanilloid ◽  
Solvent Exposure ◽  
Channel Activation ◽  
Trpv1 Channels

ABSTRACTTransient Receptor Potential Vanilloid (TRPV1) channels support the detection and integration of nociceptive input. Currently available functional and structural data suggest that that TRPV1 channels have two potential gates within their cation selective permeation pathway: a barrier formed by a ‘bundle crossing’ at the intracellular entrance and a second constriction created by the ion selectivity filter. To describe conformational changes associated with channel gating within the pore, the fluorescent non-canonical amino acid (f- ncAA) coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. TRPV1 channels expressing coumarin at either site displayed normal voltage- and agonist-dependent gating. Next, total internal reflection microscopy (TIRF) was performed to enable ultra-rapid, millisecond imaging of the conformational dynamics in single TRPV1 channels in live cells. Here, the data obtained from channels expressed in human derived cells show that optical fluctuations, photon counts, and variance of noise analysis from Y671 coumarin encoded in TRPV1 tetramers correlates closely with channel activation by capsaicin, thus providing an direct optical marker of channel activation at the selectivity filter. In companion molecular dynamics simulations, Y671 displays alternating solvent exposure between the closed and open states, giving support to the optical data. These calculations further suggest a direct involvement of Y671 in controlling the relative position of the pore helix and its role in supporting ionic conductance at the TRPV1 selectivity filter.

scholarly journals Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Ximena Steinberg ◽  
Marina A Kasimova ◽  
Deny Cabezas-Bratesco ◽  
Jason D Galpin ◽  
Ernesto Ladron-de-Guevara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Conformational Dynamics ◽  
Structural Data ◽  
Selectivity Filter ◽  
Live Cells ◽  
Solvent Exposure ◽  
Trpv1 Channels ◽  
Canonical Amino Acid ◽  
Dynamics Simulations

TRPV1 channels support the detection of noxious and nociceptive input. Currently available functional and structural data suggest that TRPV1 channels have two gates within their permeation pathway: one formed by a ′bundle-crossing′ at the intracellular entrance and a second constriction at the selectivity filter. To describe conformational changes associated with channel gating, the fluorescent non-canonical amino acid coumarin-tyrosine was genetically encoded at Y671, a residue proximal to the selectivity filter. Total internal reflection fluorescence microscopy was performed to image the conformational dynamics of the channels in live cells. Photon counts and optical fluctuations from coumarin encoded within TRPV1 tetramers correlates with channel activation by capsaicin, providing an optical marker of conformational dynamics at the selectivity filter. In agreement with the fluorescence data, molecular dynamics simulations display alternating solvent exposure of Y671 in the closed and open states. Overall, the data point to a dynamic selectivity filter that may serve as a gate for permeation.

scholarly journals Permeability Properties of Enac Selectivity Filter Mutants

2001 ◽  
Vol 118 (6) ◽  
pp. 679-692 ◽  
Author(s):  
Stephan Kellenberger ◽  
Muriel Auberson ◽  
Ivan Gautschi ◽  
Estelle Schneeberger ◽  
Laurent Schild
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Ion Selectivity ◽  
Critical Role ◽  
Selectivity Filter ◽  
Side Chain ◽  
Na Channel ◽  
Ionic Selectivity ◽  
Channel Pore ◽  
Subunit Interface

The epithelial Na+ channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na+ absorption. The amiloride-sensitive ENaC is highly selective for Na+ and Li+ ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the αS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K+ ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted αS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position α589, indicating that αS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na+ and Li+. These findings demonstrate the critical role of the pore size at the αS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the αS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the αS589 side chain is oriented toward the subunit–subunit interface and that substitution of αS589 by larger residues increases the pore diameter by adding extra volume at the subunit–subunit interface.

scholarly journals The ion selectivity filter is not an activation gate in TRPV1-3 channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrés Jara-Oseguera ◽  
Katherine E Huffer ◽  
Kenton J Swartz
Keyword(s):  
Sensory Neurons ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Ion Permeation ◽  
Structural Rearrangements ◽  
Trpv1 Channels ◽  
Activation Gate ◽  
Reactive Ions

Activation of TRPV1 channels in sensory neurons results in opening of a cation permeation pathway that triggers the sensation of pain. Opening of TRPV1 has been proposed to involve two gates that appear to prevent ion permeation in the absence of activators: the ion selectivity filter on the external side of the pore and the S6 helices that line the cytosolic half of the pore. Here we measured the access of thiol-reactive ions across the selectivity filters in rodent TRPV1-3 channels. Although our results are consistent with structural evidence that the selectivity filters in these channels are dynamic, they demonstrate that cations can permeate the ion selectivity filters even when channels are closed. Our results suggest that the selectivity filters in TRPV1-3 channels do not function as activation gates but might contribute to coupling structural rearrangements in the external pore to those in the cytosolic S6 gate.

A Review on Potential Involvement of TRPV1 Channels in Ischemia–Reperfusion Injury

2017 ◽  
Vol 23 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Puneet Kaur Randhawa ◽  
Amteshwar Singh Jaggi
Keyword(s):  
Reperfusion Injury ◽  
Inflammatory Cytokines ◽  
Transient Receptor Potential ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Channel Activation ◽  
Trpv1 Channels ◽  
Transient Receptor

Besides functioning as thermosensors, transient receptor potential vanilloid 1 (TRPV1) channels play a pivotal role in ischemia–reperfusion injury. Transient receptor potential vanilloid 1 channel activation attenuates ischemia–reperfusion-induced injury in various organs including the heart, lungs, kidneys, and the brain. Transient receptor potential vanilloid 1 channels are expressed on the sensory neurons innervating the myocardium, ventricles of the heart, epicardial surface of the heart, endothelial cells, and the vascular smooth muscle cells. During ischemic conditions, activation of TRPV1 channels on the perivascular nerves stimulates the release of calcitonin gene-related peptide and substance P to produce cardioprotection. Furthermore, TRPV1 channel activation reduces the generation of free radicals and inflammatory cytokines, inhibits neutrophil infiltration, and enhances the production of anti-inflammatory cytokines to reduce ischemia–reperfusion-induced tissue injury. The present review describes the potential involvement of TRPV1 channels and the signaling cascade in attenuating ischemia–reperfusion injury in various organs.

scholarly journals Amino Acid Substitutions in Putative Selectivity Filter Regions III and IV in KdpA Alter Ion Selectivity of the KdpFABC Complex from Escherichia coli

2004 ◽  
Vol 186 (16) ◽  
pp. 5519-5522 ◽  
Author(s):  
Jessica Bertrand ◽  
Karlheinz Altendorf ◽  
Marc Bramkamp
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Potassium Channels ◽  
Sequence Homology ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Amino Acid Substitutions ◽  
High Osmolality ◽  
Potassium Binding ◽  
Potassium Limitation

ABSTRACT When grown under conditions of potassium limitation or high osmolality, Escherichia coli synthesizes the K+-translocating KdpFABC complex. The KdpA subunit, which has sequence homology to potassium channels of the KcsA type, has been shown to be important for potassium binding and transport. Replacement of the glycine residues in KdpA at positions 345 and 470, members of putative selectivity filter regions III and IV, alters the ion selectivity of the KdpFABC complex.

scholarly journals Author response: Conformational dynamics in TRPV1 channels reported by an encoded coumarin amino acid

2017 ◽  
Author(s):  
Ximena Steinberg ◽  
Marina A Kasimova ◽  
Deny Cabezas-Bratesco ◽  
Jason D Galpin ◽  
Ernesto Ladron-de-Guevara ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Dynamics ◽  
Author Response ◽  
Trpv1 Channels ◽  
Coumarin Amino Acid

Emulsified Isoflurane Enhances Thermal Transient Receptor Potential Vanilloid-1 Channel Activation–mediated Sensory/Nociceptive Blockade by QX-314

2014 ◽  
Vol 121 (2) ◽  
pp. 280-289 ◽  
Author(s):  
Cheng Zhou ◽  
Peng Liang ◽  
Jin Liu ◽  
Wensheng Zhang ◽  
Daqing Liao ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Heat Exposure ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Trpv1 Channel ◽  
Channel Activation ◽  
Trpv1 Channels ◽  
Temperature Thresholds ◽  
Dorsal Root Ganglia Neurons ◽  
Transient Receptor

Abstract Background: QX-314 produces nociceptive blockade, facilitated by permeation through transient receptor potential vanilloid-1 (TRPV1) channels. TRPV1 channel can be activated by noxious heat and sensitized by volatile anesthetics. The authors hypothesized that emulsified isoflurane (EI) could enhance thermal TRPV1 channel activation–mediated sensory/nociceptive blockade by QX-314. Methods: Rats were perineurally injected with QX-314 (Sigma-Aldrich Co. Ltd. Shanghai, China) alone or QX-314 combined with EI, followed by heat exposure on the injection site. The tail-flick and tail-clamping tests were used to assess sensory and nociceptive blockade, respectively; a sciatic nerve block model was used to assess motor and sensory blockade. Effects of EI on thermal activation of TRPV1 channels were evaluated on rat dorsal root ganglia neurons by whole-cell patch-clamp recordings. Results: Heat exposure enhanced sensory/nociceptive blockade by QX-314 in rat tails, but not motor blockade in sciatic nerve block model. QX-314 alone or QX-314 + 42°C produced no nociceptive blockade. QX-314 + 48°C produced 100% nociceptive blockade with duration of 12.5 ± 2.0 h (mean ± SEM). By adding 2% EI, QX-314 + 42°C produced 80% nociceptive blockade with duration of 8.1 ± 1.9 h, which was similar to the effect of QX-314 + 46°C (7.7 ± 1.1 h; P = 0.781). The enhancement of heat on sensory/nociceptive blockade of QX-314 was prevented by TRPV1 channel antagonist. The temperature thresholds of TRPV1 channel activation on dorsal root ganglia neurons were significantly reduced by EI. Conclusions: Thermal activation of TRPV1 channels enhanced long-lasting sensory/nociceptive blockade by QX-314 without affecting motor blockade. The addition of EI reduced temperature thresholds for inducing long-lasting sensory/nociceptive blockade due to QX-314.

scholarly journals Modulation of the Shaker K+Channel Gating Kinetics by the S3–S4 Linker

2000 ◽  
Vol 115 (2) ◽  
pp. 193-208 ◽  
Author(s):  
Carlos Gonzalez ◽  
Eduardo Rosenman ◽  
Francisco Bezanilla ◽  
Osvaldo Alvarez ◽  
Ramon Latorre
Keyword(s):  
Amino Acid ◽  
Noise Analysis ◽  
Ionic Currents ◽  
K Channel ◽  
Total Charge ◽  
Maximum Probability ◽  
Channel Activation ◽  
Deactivation Kinetics ◽  
The Right ◽  
S4 Segment

In Shaker K+ channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3–S4 linker. To investigate the role of the S3–S4 linker in the ShakerH4Δ(6–46) (ShakerΔ) K+ channel activation, we constructed S3–S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K+ currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerΔ. The apparent number of gating charges was 12.6 ± 0.6 eo in ShakerΔ, 12.7 ± 0.5 in 10 aa linker, and 12.3 ± 0.9 in 5 aa linker channels, but it was only 5.6 ± 0.3 eo in the 0 aa linker mutant channel. The maximum probability of opening (Pomax) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89× and 45× slower than that of the ShakerΔ K+ channel, respectively. The initial lag of ionic currents when the prepulse was varied from −130 to −60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3–S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3–S4 linker plays an important role in setting ShakerΔ channel activation and deactivation kinetics.

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