Biology subjects

Ruehl, P.

Publications and source records attributed to Ruehl, P..

4 recordsLinked to original sources

bioRxiv2026

Structural compatibility enables functional co-assembly of Kir2.1 channels and the voltage-sensing domain of voltage-sensitive phosphatases

Voltage-sensing domains (VSDs), which are integral parts of voltage-gated K+ channel (KV) proteins, are highly modular protein components that function largely independently of ion-conducting pores - a property exploited in genetically encoded voltage indicators (GEVIs). Conversely, inward-rectifier K+ channels such as Kir2.1 possess a pore-only architecture and lack a VSD. Here, we demonstrate an unexpected and functionally relevant structural compatibility between the independently evolved pore-only and VSD-only membrane protein families. When co-expressed, Kir2.1 and ASAP-type GEVIs form complexes that constrain VSD movement and markedly interfere with voltage-dependent fluorescence responses. Molecular modeling combined with targeted mutagenesis identified a conserved hydrophobic interface that mediates this interaction. A single bulky substitution in the VSD of the GEVI rEstus-NI (A79W) disrupted the impact of Kir2.1 while preserving the GEVIs voltage-sensing performance. These findings suggest that pore-only and VSD-only proteins can assemble into functional KV-like architectures, and highlight that membrane proteins may engage in unexpected interactions capable of altering experimental readouts in physiological voltage imaging studies. The study also raises the possibility that independently functional membrane proteins may assemble into previously unrecognized higher-order complexes with distinct functional properties under native physiological conditions.

physiology↗

bioRxiv2026

Sub-Millivolt Voltage Imaging Reveals Gap Junction-Mediated Bioelectric Contact Inhibition

Sub-millivolt membrane potential (Vm) dynamics in multicellular non-excitable networks have remained largely invisible due to a lack of sufficiently sensitive imaging tools. Here, we introduce rEstus2s, a next-generation genetically encoded voltage indicator that overcomes this barrier by enabling high-resolution Vm imaging across the full physiological resting Vm range (-100 to 0 mV). Using rEstus2s, we uncover bioelectric contact inhibition (BCI), a fundamental biophysical principle where gap junction coupling acts as a passive noise filter to stabilize Vm. We demonstrate that the time-dependent variance of Vm (electrical volatility) relative to the number of cells (n) in a network follows a 1/n scaling law, reflecting a transition from stochastic single-cell behavior to collective electrical stability. While Ca2+-activated oncogenic ion channels, including ANO1 and KCa3.1, promote pronounced electrical volatility in isolated cells, BCI effectively attenuates volatility in electrically coupled networks. Disruption of gap junction coupling in cancer cells abolishes BCI and restores high electrical volatility. These findings establish a unifying biophysical framework for understanding how multicellularity maintains electrical homeostasis in health and disease. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/701308v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@120fcb9org.highwire.dtl.DTLVardef@affa2aorg.highwire.dtl.DTLVardef@150ffa7org.highwire.dtl.DTLVardef@c05392_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Absolute Membrane Potential Recording with ASAP-Type Genetically Encoded Voltage Indicators Using Fluorescence Lifetime Imaging

The electrical membrane voltage (Vm) characterizes the functional state of biological cells, thus requiring precise, non-invasive Vm-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate Vm changes, variability in sensor expression confound determination of absolute Vm. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. With the highest lifetime change in the Vm range of -100 to 50 mV of about 730 ps, ASAP3 and rEstus-NI are preferred for FLIM recordings. At a physiologically relevant Vm of -30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting Vm in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous Vm fluctuations in A375 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/669310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eaff8borg.highwire.dtl.DTLVardef@12f1a54org.highwire.dtl.DTLVardef@169ad83org.highwire.dtl.DTLVardef@fe9f44_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

An ultrasensitive genetically encoded voltage indicator uncovers the electrical activity of non-excitable cells

Genetically encoded voltage indicators (GEVIs) are powerful, non-invasive tools for recording action potentials in excitable cells. However, most animal cell types are non-excitable, and yet variations in the membrane potential are biologically relevant in these cells as well. Resolving such small voltage signals demands GEVIs with exceptionally high sensitivity. In this study, we applied structure-guided engineering to the GEVI ASAP3 to generate rEstus, a sensor with optimized brightness, voltage sensitivity, and voltage range. rEstus is most sensitive in the resting voltage range of non-excitable cells, exhibits a 3.6-fold improvement in fast voltage spike detection, and allows for absolute voltage calibration at the single-cell level. Using rEstus, we resolved endogenous voltage fluctuations in several non-excitable cell types and demonstrate that correlation analysis of these optically recorded fluctuations provides an easy, non-invasive, real-time readout of electrical gap-junction coupling. Our work provides greatly enhanced tools and methods for the non-invasive study of electrical signaling in excitable and non-excitable cells.

biophysics↗