Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels

Indexación: Scopus.ACKNOWLEDGMENTS. We thank Chris Lingle and Yu Zhou (Washington University) for critical reading of the manuscript and Victoria Prado for Xenopus care and oocyte preparation. We also thank Millennium Scientific Initiative P029-022-F. This work was supported by Fondecyt Postdoctoral Grants 3170599 (to I.D.-F.) and 3160321 (to H.M.).Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1–R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum. © National Academy of Sciences. All rights reserved.https://www.pnas.org/content/115/32/820

Novel TRPV1 Channel Agonists With Faster and More Potent Analgesic Properties Than Capsaicin

Indexación: Scopus.The transient receptor potential vanilloid 1 (TRPV1) ion channel is a member of the family of Transient Receptor Potential (TRP) channels that acts as a molecular detector of noxious signals in primary sensory neurons. Activated by capsaicin, heat, voltage and protons, it is also well known for its desensitization, which led to the medical use of topically applied TRPV1 agonist capsaicin for its long-lasting analgesic effects. Here we report three novel small molecules, which were identified using a Structure-Based Virtual Screening for TRPV1 from the ZINC database. The three compounds were tested using electrophysiological assays, which confirmed their capsaicin-like agonist activity. von Frey filaments were used to measure the analgesic effects of the compounds applied topically on tactile allodynia induced by intra-plantar carrageenan. All compounds had anti-nociceptive activity, but two of them showed faster and longer lasting analgesic effects than capsaicin. The present results suggest that TRPV1 agonists different from capsaicin could be used to develop topical analgesics with faster onset and more potent effects. © Copyright © 2020 Duarte, Cáceres, Sepúlveda, Arriagada, Olivares, Díaz-Franulic, Stehberg and González-Nilo.https://www.frontiersin.org/articles/10.3389/fphar.2020.01040/ful

Pore size matters for potassium channel conductance

Ion channels are membrane proteins that mediate efficient ion transport across the hydrophobic core of cell membranes, an unlikely process in their absence. K+ channels discriminate K+ over cations with similar radii with extraordinary selectivity and display a wide diversity of ion transport rates, covering differences of two orders of magnitude in unitary conductance. The pore domains of large- and small-conductance K+ channels share a general architectural design comprising a conserved narrow selectivity filter, which forms intimate interactions with permeant ions, flanked by two wider vestibules toward the internal and external openings. In large-conductance K+ channels, the inner vestibule is wide, whereas in small-conductance channels it is narrow. Here we raise the idea that the physical dimensions of the hydrophobic internal vestibule limit ion transport in K+ channels, accounting for their diversity in unitary conductance

Structure-driven pharmacology of transient receptor potential channel vanilloid 1

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal receptor that mediates the flux of cations across the membrane in response to several stimuli, including heat, voltage, and ligands. The best known agonist of TRPV1 channels is capsaicin, the pungent component of “hot” chili peppers. In addition, peptides found in the venom of poisonous animals, along with the lipids phosphatidylinositol 4,5-biphosphate, lysophosphatidic acid, and cholesterol, bind to TRPV1 with high affinity to modulate channel gating. Here, we discuss the functional evidence regarding ligand-dependent activation of TRPV1 channels in light of structural data recently obtained by cryoelectron microscopy. This review focuses on the mechanistic insights into ligand binding and allosteric gating of TRPV1 channels and the relevance of accurate polymodal receptor biophysical characterization for drug design in novel pain therapies

K+ Conduction and Mg2+ Blockade in a Shaker Kv-Channel Single Point Mutant with an Unusually High Conductance

Vergara-Jaque, A (Vergara-Jaque, Ariela). Univ Talca, Ctr Bioinformat & Simulac Mol, Talca, ChilePotassium channels exhibit a large diversity of single-channel conductances. Shaker is a low-conductance K-channel in which Pro475 -> Asp, a single-point mutation near the internal pore entrance, promotes 6- to 8-fold higher unitary current. To assess the mechanism for this higher conductance, we measured Shaker-P475D single-channel current in a wide range of symmetrical K+ concentrations and voltages. Below 300 mM K+, the current-to-voltage relations (i-V) showed inward rectification that disappeared at 1000 mM K+. Single-channel conductance reached a maximum of similar to 190 pS at saturating [K+], a value 4- to 5-fold larger than that estimated for the native channel. Intracellular Mg2+ blocked this variant with similar to 100-fold higher affinity. Near zero voltage, blockade was competitively antagonized by K+; however, at voltages >100 mV, it was enhanced by K+. This result is consistent with a lock-in effect in a single-file diffusion regime of Mg2+ and K+ along the pore. Molecular-dynamics simulations revealed higher K+ density in the pore, especially near the Asp-475 side chains, as in the high-conductance MthK bacterial channel. The molecular dynamics also showed that K+ ions bound distally can coexist with other K+ or Mg2+ in the cavity, supporting a lock-in mechanism. The maximal K+ transport rate and higher occupancy could be due to a decrease in the electrostatic energy profile for K+ throughout the pore, reducing the energy wells and barriers differentially by similar to 0.7 and similar to 2 kT, respectively