Biology subjects

Lacroix, J. J.

Publications and source records attributed to Lacroix, J. J..

11 recordsLinked to original sources

bioRxiv2026

Yoda molecules agonize PIEZO2

PIEZO proteins (PIEZO1 and PIEZO2) are essential mechanosensitive channels. PIEZO1 is thought to be selectively activated by Yoda molecules (Yoda1 and Yoda2). Although a structural framework for PIEZO1 activation by Yoda1 exists, a molecular mechanism underlying this selective activation is lacking. Here, using electrophysiology and calcium imaging, we show that Yoda1 increases PIEZO2 open probability and stretch sensitivity as efficaciously as PIEZO1 but elicits weaker PIEZO2-dependent calcium entry, rationalizing why its effect on PIEZO2 has been overlooked. Both Yoda1 and its more potent Yoda2 analog slow down inactivation of PIEZO2 currents with potency similar to PIEZO1 but with lower efficacy. Using mutagenesis and molecular dynamics simulations, we further show that Yoda2s benzoic acid group forms a transient salt bridge with a conserved arginine in the Yoda binding site, providing a molecular basis for Yoda2s increased potency. Our study cautions a reevaluation of studies using these molecules to untangle biological functions mediated by PIEZO channels.

biophysics↗

bioRxiv2024

Dissecting current rectification through asymmetric nanopores

Rectification, the tendency of bidirectional ionic conductors to favor ion flow in a specific direction, is an intrinsic property of many ion channels and synthetic nanopores. Despite its frequent occurrence in ion channels and its phenomenological explanation using Eyrings rate theory, a quantitative relationship between the rectified current and the underlying ion-specific and voltage-dependent free energy profile has been lacking. In this study, we designed nanopores in which potassium and chloride current rectification can be manipulated by altering the electrostatic pore polarity. Using molecular dynamics-based free energy simulations, we quantified voltage-dependent changes of free energy barriers in six ion-nanopore systems. Our results illustrate how the energy barriers for inward and outward fluxes become unequal in the presence of an electromotive driving force, leading to varying degrees of rectification for cation and anion currents. This work establishes a direct link between equilibrium potential of mean force and current rectification rate and demonstrates that rectification caused by energy barrier asymmetry depends on the nature of the permeating ion, can be tuned by pore polarity, does not require ion binding sites, conformational flexibility, or specific pore geometry, and, as such, may be widespread among ion channels. Statement of significanceIn many ion channels, ions flow faster in one direction than the other, even under equal magnitude of driving forces. This phenomenon, known as rectification, often arises as an intrinsic biophysical property of the channel pore and has significant physiological implications. Our molecular dynamics simulations provide a unified framework to dissect the free energy profiles that underpin current rectification. This approach has broad applications in the field of ion channel biology and material design.

biophysics↗

bioRxiv2024

Mammalian PIEZO channels rectify anionic currents

Under physiological conditions, mammalian PIEZO channels (PIEZO1 and PIEZO2) elicit transient currents mostly carried by monovalent and divalent cations. PIEZO1 is also known to permeate chloride ions, with a Cl- / Na+ permeability ratio of about 0.2. Yet, little is known about how anions permeate PIEZO channels. Here, by separately measuring sodium and chloride currents using non-permanent counter-ions, we show that both PIEZO1 and PIEZO2 rectify chloride currents outwardly, favoring entry of chloride ions at voltages above their reversal potential, whereas little to no rectification was observed for sodium currents. Interestingly, chloride currents elicited by 9K, an anion-selective PIEZO1 mutant harboring multiple positive residues along intracellular pore fenestrations, also rectify but in the inward direction. Molecular dynamics simulation indicate that the inward rectification of chloride currents in 9K correlates with the largely positive electrostatic potential at the intracellular pore entrance, suggesting that rectification can be tuned by pore polarity. These results demonstrate that the pore of mammalian PIEZO channels inherently rectifies chloride currents. Statement of significanceMechanosensitive PIEZO ion channels play many important roles across cells and tissues. Their open pore facilitates the flow of cations down their electrochemical gradients, eliciting sodium-driven membrane depolarization and calcium-dependent signaling under physiological conditions. Yet, these channels also permeate chloride ions. In this study, we show that the two mammalian PIEZO channel homologs preferentially permeate chloride ions into the cells at voltages more positive than the chloride reversal potential. Although PIEZOs permeate cations more effectively than chloride ions, the influx of chloride ions mediated by PIEZOs could participate in certain physiological processes.

biophysics↗

bioRxiv2022

Binding Free Energies of Piezo1 Channel Agonists at Protein-Membrane Interface

Mechanosensitive Piezo channels convert mechanical stimuli into biological signals in vertebrates. Piezo1 chemical modulators are anticipated to yield many clinical benefits. To date, Yoda1 is the most potent and widely used Piezo1-selective agonist, yet how Yoda1 interacts with Piezo1 at the protein-membrane interface and stabilizes Piezo1s open state remains elusive. Here, using a previously identified putative Yoda1 binding site and three molecular dynamics (MD)-based methods, we computed the binding free energies of Yoda1 and its analogs in a Piezo1 cryo-EM closed state and an in silico open state. Our computed absolute binding free energy of Yoda1 in the closed state agrees well with the experimental Kd in which Piezo1 is expected to be in a closed state. More importantly, Yoda1 binds the open state better than the closed state, in agreement with its agonist effects. All three methods predicted that Dooku1, a Yoda1 analog, binds the closed state stronger than Yoda1, but binds the open state weaker than Yoda1. These results are consistent with the fact that Dooku1 antagonizes the effects of Yoda1 but lacks the ability to activate Piezo1. The relative binding free energies of seven Yoda1 analogs recapitulate key experimental structure-activity-relationships (SAR). Based on the state-dependent binding free energies, we were able to predict whether a molecule is an agonist or inhibitor and whether a chemical modification will lead to a change in affinity or efficacy. These mechanistic insights and computational workflow designed for transmembrane binders open an avenue to structural-based screening and design of novel Piezo1 agonists and inhibitors.

biophysics↗

bioRxiv2022

Rearrangements of Piezo1 blade domains correlate with pore opening

PIEZO1 channels open in response to numerous mechanical stimuli, such as physical membrane deformations, which modulate the curvature of flexible domains called blades. Yet, whether different stimuli cause similar blade motions and whether these rearrangements correlate with pore opening remain unclear. Here, we scan local conformational changes along the PIEZO1 blade using fluorescent probes. We identify two distant probes, one intracellular proximal and the other extracellular distal, which independently and robustly respond to flow stimuli. Flow-induced signals from both probes exquisitely correlate with PIEZO1-dependent calcium influx and specifically increase in presence of fast-inactivating pore mutations. In contrast, both probes remain fluorimetrically silent to hypotonic shocks and indentations, two stimuli that otherwise evoke normal electrochemical responses in both engineered channels. This study reveals that flow-induced blade motions are functionally coupled to the pore and that at least two distant blade regions discriminate flow from two other stimuli, suggesting that PIEZO1 mobilizes distinct mechanisms to sense a broad range of mechanical cues. TeaserFluorimetric evidence suggests that different mechanical stimuli impart distinct rearrangements in PIEZO1s mechanosensory domains.

biophysics↗

bioRxiv2021

Lipids in the Piezo1 channel pore: gating mechanism, or simulation artifact?

Multiscale molecular dynamics (MD) simulations using Martini coarse-grained (CG) and all-atom (AA) forcer fields are commonly used in membrane protein studies. In particular, reverse-mapping an equilibrated CG model to an AA model offers an efficient way for preparing large membrane protein systems with complex protein shapes and lipid compositions. Here, we report that this hybrid CG-equilibrium-AA-production protocol may artificially increase lipid density and decrease hydration in ion channel pores walled with transmembrane gaps. To understand the origin of this conundrum, we conducted replicas of CG, AA, and reverse-mapped AA simulations of the pore domain of the mechanosensitive Piezo1 channel in a non-conducting conformation. Lipid/water density analysis and free energy calculations reveal that the lack of initial pore hydration allows adjacent lipids to enter the pore lumen through gaps between pore helices during CG simulation. Due to the mismatch between CG and AA lipid kinetics, these pore lipids remain trapped in the subsequent AA simulations, despite unfavorable binding free energy. We tested several CG equilibrium protocols and found that a protocol restraining the whole lipid produces pore hydration consistent with AA results, thus eliminating this artifact for further studies of lipid gating and protein-lipid interactions. WHY IT MATTERSMembrane-embedded proteins constantly interact with lipid molecules. Computational molecular dynamics simulations have become an indispensable tool for investigating the role of such protein-lipid interactions. Using mechanosensitive Piezo1 channel as model, we found that subtle differences in solvation and equilibrium protocols between coarse-grained and all-atom MD simulations can result in different lipid densities inside the channel pore. We identify the underlying cause of this discrepancy and propose alternative protocols to avoid this artifact.

biophysics↗

bioRxiv2021

An inter-channel cooperative mechanism mediates PIEZO1's exquisite mechanosensitivity

The bowl-shaped structure of PIEZO channels is predicted to flatten in response to mechanical stimuli, gating their pore open. However, how this unique structure allows them to detect exquisitely small changes in membrane tension remains unclear. Here, using pressure clamp electrophysiology, modeling, and molecular dynamics simulations, we show that the single channel open probability of PIEZO1 increases weakly with respect to pressure-induced tension. In contrast, when multiple channels are present in a membrane patch, channel open probability increases steeply as a function of the number of open channels. These cooperative effects are consistent with an inter-channel energetic repulsion due to the local membrane deformation created by the non-planar PIEZO structure. When channels open, this deformation shrinks, allowing open channels to diffuse closer to each other, thus delaying closure. This study reveals how PIEZO1 channels acquire their exceptional mechanosensitivity and suggests a possible mechanism by which cells could rapidly tune mechanosensitivity.

biophysics↗

bioRxiv2020

Focused ultrasound excites neurons via mechanosensitive calcium accumulation and ion channel amplification

Ultrasonic neuromodulation has the unique potential to provide non-invasive control of neural activity in deep brain regions with high spatial precision and without chemical or genetic modification. However, the biomolecular and cellular mechanisms by which focused ultrasound excites mammalian neurons have remained unclear, posing significant challenges for the use of this technology in research and potential clinical applications. Here, we show that focused ultrasound excites neurons through a primarily mechanical mechanism mediated by specific calcium-selective mechanosensitive ion channels. The activation of these channels results in a gradual build-up of calcium, which is amplified by calcium- and voltage-gated channels, generating a burst firing response. Cavitation, temperature changes, large-scale deformation, and synaptic transmission are not required for this excitation to occur. Pharmacological and genetic inhibition of specific ion channels leads to reduced responses to ultrasound, while over-expressing these channels results in stronger ultrasonic stimulation. These findings provide a critical missing explanation for the effect of ultrasound on neurons and facilitate the further development of ultrasonic neuromodulation and sonogenetics as unique tools for neuroscience research.

neuroscience↗

bioRxiv2020

Multimodal activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter

G-protein coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and mechanical stimuli to G protein signaling and plays important roles in vascular physiology, neuroplasticity and cancer progression. Here, we designed a genetically-encoded fluorescent reporter of GPR68 activation called "iGlow". iGlow responds to known GPR68 activators including fluid shear stress, extracellular pH and the synthetic agonist ogerin. Remarkably, iGlow activation occurred from both primary cilia-like structures and from intracellular vesicles, showing iGlow senses extracellular flow from within the cell. Flow-induced iGlow activation is not eliminated by pharmacological modulation of G protein signaling, disruption of actin filaments, or the presence of GsMTx4, a non-specific inhibitor of mechanosensitive ion channels. Genetic deletion of the conserved Helix 8, proposed to mediate GPCR mechanosensitivity, did not eliminate flow-induced iGlow activation, suggesting GPR68 uses a hitherto unkonwn, Helix8-independent mechanism to sense mechanical stimuli. iGlow will be useful to investigate the contribution of GPR68-mediated mechanotransduction in health and diseases.

biophysics↗

bioRxiv2020

A Piezo1 open state reveals a multi-fenestrated ion permeation pathway

Force-sensing Piezo channels are essential to many aspects of vertebrate physiology. Activation of Piezo1 is facilitated by the presence of negative membrane lipids in the inner leaflet, such as phosphatidylinositol-4,5-bisphosphate (PIP2). Here, to study how Piezo1 opens, we performed molecular dynamics simulations of Piezo1 in membranes flattened by the periodic boundary effect and with or without PIP2 lipids. The Piezo1 pore spontaneously opens in the asymmetrical bilayer but not in the symmetric membrane or when PIP2 lipids are neutralized. Electrophysiological characterization of putative PIP2-interacting Piezo1 residues suggests the contribution of multiple PIP2 binding sites. Our Piezo1 open state recapitulates ionic selectivity, unitary conductance and mutant phenotypes obtained from numerous experimental studies. Tracking ion diffusion through the open pore reveals the presence of intracellular and extracellular fenestrations, delineating a multi-fenestrated permeation pathway. This open state sheds light on the mechanisms of lipid modulation, permeation, and selectivity in a Piezo channel.

biophysics↗

bioRxiv2017

Chemical Gating of the Mechanosensitive Piezo1 Channel by Asymmetric Binding of its Agonist Yoda1

Piezo proteins are homotrimeric ion channels that play major roles in normal and pathological mechanotransduction signaling in mammalian organisms. Their pharmacological control hence represents a potential therapeutic avenue. Yoda1, a Piezo1-selective small molecule agonist, is the only known selective Piezo modulator. How Yoda1 selectively interacts with Piezo1 and opens its pore is unknown. Here, by engineering and characterizing chimeras, we identified a minimal region responsible for Yoda1 binding. This region is located at the interface between the pore and the putative mechanosensory domains in each subunit. By characterizing hybrid channels containing Yoda1-insensitive and Yoda1-sensitive monomers, we demonstrate that the presence of only one Yoda1-sensitive Piezo1 subunit is sufficient for chemical activation, implicating that the asymmetric binding of Yoda1 to a single subunit enables channel opening. These findings shed light onto the gating mechanisms of Piezo channels and will pave the way for the rationale design of new Piezo channels modulators.

biochemistry↗