Biology subjects

Verkest, C.

Publications and source records attributed to Verkest, C..

6 recordsLinked to original sources

bioRxiv2026

State-dependent binding of the wedge domain controls inactivation of the mechanosensitive ion channel PIEZO1

The mechanically activated ion channel PIEZO1 transduces membrane tension into intracellular calcium signals and is critical for a wide range of physiological processes. Recent structural and functional studies have established a detailed framework for PIEZO1 activation, but the molecular mechanisms governing its rapid inactivation remain incompletely understood. Here, we examined the contribution of the intracellular wedge domain to PIEZO1 inactivation using site-directed mutagenesis, electrophysiological recordings and MINFLUX nanoscopy. We show that wedge deletion and disruption of specific {pi}-{pi} and cation-{pi} interactions between the wedge 1-helix and the pore module abolishes inactivation without impairing channel activation. Moreover, MINFLUX nanoscopy reveals that the wedge stabilizes a flat inactivated conformation of PIEZO1 and suggests that wedge dissociation is required for recovery from inactivation. Together, our data support a ball-and-chain-like mechanism with the wedge acting as a state-dependent inactivation particle that docks to the pore module to terminate channel activity during sustained mechanical stimulation.

physiology↗

bioRxiv2025

3D-MINFLUX nanoscopy reveals distinct allosteric mechanisms for activation and modulation of PIEZO1 by Yoda1

PIEZO1 underpins numerous physiological processes by which cells detect and respond to mechanical stimuli. The small molecule Yoda1 has become an indispensable tool that is frequently used for dissecting the role of PIEZO1 in physiological and pathological contexts, yet its mode of action remains incompletely understood. Here, using site-directed mutagenesis, electrophysiology, computational modelling and 3D-MINFLUX nanoscopy, we demonstrate that mutation of the previously proposed Yoda1 binding site, solely abolishes Yoda1-induced activation and channel flattening but preserves modulation of mechanically-evoked PIEZO1 currents, whereas mutation of F1715, which lines a transient binding cavity accessible only in the flattened PIEZO1 conformation, eliminated modulation without affecting Yoda-induced calcium entry. Thus, our data support a two-site induced-fit-like mechanism where Yoda first engages binding site-1 to promote blade flattening and pore opening and then transitions to a deeper binding site to modulate the mechanical activation threshold and inactivation kinetics. This revised model distinguishes discrete allosteric pathways for PIEZO1 activation versus modulation and provides a framework for the design of next-generation use-dependent PIEZO1 modulators.

pharmacology and toxicology↗

bioRxiv2024

Cluster nanoarchitecture and structural diversity of PIEZO1 in intact cells

The force-gated ion channel PIEZO1 confers mechanosensitivity to many cell types. While the structure and physiological roles of PIEZO1 are well-described, the subcellular distribution and the impact of the cellular microenvironment on PIEZO1 conformation and function are poorly understood. Here, using MINFLUX nanoscopy we demonstrate that PIEZO1 channels collectively deform the membrane into pit-shaped invaginations, thereby creating mechano-responsive microdomains capable of amplifying mechanical stimuli via subtle changes in their topology. Moreover, by measuring intramolecular distances in individual PIEZO1 channels with nanometer precision, we reveal subcellular compartment-specific differences in PIEZO1 conformation at rest and during activation that correlate with differences in PIEZO1 function and are possibly caused by differences in cytoskeletal architecture. Together, our data provide previously unrecognized insights into the complex interplay of forces that determine how PIEZO1 alters membrane shape and, vice versa, how the membrane together with the cytoskeleton affect the conformation and function of individual PIEZO1 channels. TeaserMINFLUX nanoscopy reveals subcellular distribution and conformational diversity of PIEZO1 channels in intact cells.

cell biology↗

bioRxiv2022

The molecular mechanism and physiological role of silent nociceptor activation

Silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation selectively upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors. Moreover, we show that mice lacking TMEM100 do not develop secondary allodynia - i.e. pain hypersensitivity that spreads beyond the site of inflammation - in a mouse model of knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce allodynia in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary allodynia during inflammation.

neuroscience↗

bioRxiv2021

C-Jun N-terminal kinase post-translational regulation of pain-related Acid-Sensing Ion Channels 1b and 3

Neuronal proton-gated Acid-Sensing Ion Channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathological diseases. Such conditions often involve in parallel the activation of various signaling pathways such as the Mitogen Activated Protein Kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal Kinase (JNK), as a new post-translational positive regulator of ASIC channels in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit and species dependent manner, targeting both rat and human ASIC1b and ASIC3 subunits but only mouse ASIC1b subunit. The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost upon mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNF. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons, and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.

neuroscience↗

bioRxiv2021

An intrinsically disordered intracellular domain of PIEZO2 is required for force-from-filament activation of the channel

A central question in mechanobiology is how mechanical forces acting in or on a cell are transmitted to mechanically-gated PIEZO channels that convert these forces into biochemical signals. Here we show that PIEZO2 is sensitive to force-transmission via the membrane (force-from-lipids) as well as force transmission via the cytoskeleton (force-from-filament) and demonstrate that the latter requires the intracellular linker between the transmembrane helices nine and ten (IDR5). Moreover, we show that rendering PIEZO2 insensitive to force-from-filament by deleting IDR5 abolishes PIEZO2-mediated inhibition of neurite outgrowth, which relies on the detection of cellgenerated traction forces, while it only partially affects its sensitivity to cell indentation and does not at all alter its sensitivity to membrane stretch. Hence, we propose that PIEZO2 is a polymodal mechanosensor that detects different types of mechanical stimuli via different force transmission pathways, which highlights the importance of utilizing multiple complementary assays when investigating PIEZO channel function.

neuroscience↗