Biology subjects

Jara-Oseguera, A.

Publications and source records attributed to Jara-Oseguera, A..

6 recordsLinked to original sources

bioRxiv2026

Deep mutational scan of the pore of the cold-sensing TRPM8 channel

Members of the Transient Receptor Potential (TRP) family of ion channels have a nearly ubiquitous role in human physiology, tuning cell signaling to remarkably diverse physical and chemical stimuli. Although there is extensive structural data on TRP channels, a systematic and unbiased interrogation of structure-function relations in these proteins is required to fully elucidate their mechanisms of function. By focusing on a critical pore region of the TRPM8 channel, which is the main detector of cold and cooling agents in sensory neurons, we show how deep mutational scanning can be used in combination with the available structural data to understand how TRP channels respond to stimuli. We define a novel mechanism whereby the extracellular pore loop, which has only been resolved in structures representing desensitized states of the channel, plays an essential role in the response of TRPM8 to menthol or cold by coordinating the movement of the S6 helices that line and gate the pore, and the ion-selectivity filter that binds permeant cations. Moreover, our screen reveals sequence determinants along the S6 helices that explain how their architecture sustains gating and, together, provide strong support for a structural mechanism of TRPM8 pore opening in response to menthol and cold.

biophysics↗

bioRxiv2025

Missense variant analysis in the TRPV1 ARD reveals the unexpected functional significance of a methionine

The Transient Receptor Potential Vanilloid sub-type 1 (TRPV1) is an ion channel that is activated by heat, extracellular protons, oxidation, and it is implicated in various aspects of inflammatory pain. In this study, we uncover that residue M308 in the TRPV1 ankyrin repeat domain (ARD) stands out from most other buried ARD residues because of the greater number of human missense variants at this position while maintaining a high degree of conservation across species and TRPV channel subtypes. We use mutagenesis and electrophysiology to examine this apparent discrepancy and show that substitutions at position M308 that preserve or reduce side-chain volume have no effect on channel function, whereas substitutions with larger or more polar residues increase channel activity in response to capsaicin or temperature. Substitution of M308 with a histidine bestows channels with pH-dependence that is different from wild type, consistent with the side-chain at position 308 exerting an influence on channel gating. We speculate that M308 is highly conserved because its side-chain could serve as a target for oxidation-dependent modification. On the other hand, we show that a previously described splice variant of TRPV1 that relies on M308 as a start codon diminishes surface expression of co-transfected full-length TRPV1 in HEK293 cells. Together, our findings reveal a functionally important conserved site within the ARD of TRPV1 that could have roles in oxidation-dependent channel regulation as well as tuning the number of active channels in the membrane by enabling expression of a shorter dominant-negative splice variant.

biophysics↗

bioRxiv2023

Structural dynamics at cytosolic inter-protomer interfaces control gating of a mammalian TRPM5 channel

The Transient Receptor Potential Melastatin (TRPM) family of tetrameric cation channels is involved in a wide range of biological functions, from sensory perception to cardiac function. The structurally conserved TRPM cytoplasmic domains make up > 70 % of the total protein. To investigate the mechanism by which the TRPM cytoplasmic domains contribute to gating, we employed electrophysiology and cryo-EM to study TRPM5 - a channel that primarily relies on activation via intracellular Ca2+. Here we show that activation of mammalian TRPM5 channels is strongly altered by desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca2+ binding. These transitions involve formation and dissolution of cytoplasmic inter-protomer interfaces, where the MHR1/2 domain and the rib helix play important roles. This study shows the importance of the cytoplasmic assembly in TRPM5 channel function and sets the stage for future investigations of other members of the TRPM family.

biophysics↗

bioRxiv2023

Cannabidiol sensitizes TRPV2 channels to activation by 2-APB

The cation-permeable TRPV2 channel is essential for cardiac and immune cells. Cannabidiol (CBD), a non-psychoactive cannabinoid of clinical relevance, is one of the few molecules known to activate TRPV2. Using the patch-clamp technique we discover that CBD can sensitize current responses of the rat TRPV2 channel to the synthetic agonist 2-aminoethoxydiphenyl borate (2- APB) by over two orders of magnitude, without sensitizing channels to activation by moderate (40 C) heat. Using cryo-EM we uncover a new small-molecule binding site in the pore domain of rTRPV2 that can be occupied by CBD in addition to a nearby CBD site that had already been reported. The TRPV1 and TRPV3 channels share >40% sequence identity with TRPV2 are also activated by 2-APB and CBD, but we only find a strong sensitizing effect of CBD on the response of mouse TRPV3 to 2-APB. Mutations at non-conserved positions between rTRPV2 and rTRPV1 in either the pore domain or the CBD sites failed to confer strong sensitization by CBD in mutant rTRPV1 channels. Together, our results indicate that CBD-dependent sensitization of TRPV2 channels engages multiple channel regions and possibly involves more than one CBD and 2-APB sites. The remarkably robust effect of CBD on TRPV2 and TRPV3 channels offers a promising new tool to both understand and overcome one of the major roadblocks in the study of these channels - their resilience to activation.

biophysics↗

bioRxiv2020

Global alignment and assessment of TRP channel transmembrane domain structures to explore functional mechanisms

The recent proliferation of published TRP channel structures provides a foundation for understanding the diverse functional properties of this important family of ion channel proteins. To facilitate mechanistic investigations, we constructed a structure-based alignment of the transmembrane domains of 120 TRP channel structures. Comparison of structures determined in the absence or presence of activating stimuli reveals similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available structures represent non-conducting states. Comparison of the ion selectivity filters towards the extracellular end of the pore supports existing hypotheses for mechanisms of ion selectivity. Also conserved to varying extents are hot spots for interactions with hydrophobic ligands, lipids and ions, as well as discrete alterations in helix conformations. This analysis therefore provides a framework for investigating the structural basis of TRP channel gating mechanisms and pharmacology, and, despite the large number of structures included, reveals the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function.

biophysics↗

bioRxiv2018

Conserved allosteric pathways for activation of TRPV3 revealed through engineering vanilloid-sensitivity

The Transient Receptor Potential Vanilloid 1 (TRPV) channel is activated by an array of stimuli, including heat and vanilloid compounds. The TRPV1 homologues TRPV2 and TRPV3 are also activated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. It was recently discovered that four mutations in TRPV2 are sufficient to render the channel sensitive to the TRPV1-specific vanilloid agonist resiniferatoxin (RTx). Here we show that mutation of six residues in TRPV3 corresponding to the vanilloid site in TRPV1 is sufficient to engineer RTx binding. However, robust activation of TRPV3 by RTx requires facilitation of channel opening by introducing individual mutations in the pore, temperatures > 30{degrees}C, or co-stimulation with another agonist. Our results demonstrate that the energetics of channel activation can determine the apparent sensitivity to a stimulus and suggest that allosteric pathways for activation are conserved in the TRPV family.

neuroscience↗