Biology subjects

Heinemann, S. H.

Publications and source records attributed to Heinemann, S. H..

7 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↗

bioRxiv2025

Voltage-sensor trapping of cardiac Na+ channels by Mg-protoporphyrin impairs cancer cell migration

The human voltage-gated sodium channel hNaV1.5 is essential for cardiac excitability. Though underrecognized, NaV1.5 is also expressed in multiple cancers, promoting cell migration and malignancy. hNaV1.5 is a therapeutic target but limited isoform specificity presents a risk of side effects via neuronal and skeletal muscle NaV channels. Here we identify Mg2+-protoporphyrin IX (MgPpIX), a Mg-containing tetrapyrrole and intermediate in chlorophyll biosynthesis, as inhibitor of hNaV1.5 (IC50 of 1 nM). The activity profile of various metal protoporphyrins correlates with the electrostatic potential at the metal center of the compounds. MgPpIX is specific to hNaV1.5, as no inhibition of other hNaV isoforms (hNaV1.2, 1.4, 1.7, 1.8) was detected. A mutagenesis study and structural modeling reveals that MgPpIX stabilizes the domain-II voltage sensor in the deactivated conformation, with residues E795 and N803 being relevant determinants. MgPpIX also inhibits native NaV channels in breast cancer MDA-MB-231 and colorectal carcinoma SW480 cell lines, and suppresses cell migration. MgPpIX is an exceptionally potent and specific inhibitor of hNaV1.5 and may serve as a lead compound in anti-cancer drug development.

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↗

bioRxiv2021

The liverwort Marchantia polymorpha operates a depolarization-activated Slowpoke (SLO) K+ channel that recognises pH changes in the environment

Voltage-dependent ion channels are a prerequisite for cellular excitability and electrical communication - important traits for multicellular organisms to thrive in a changeable terrestrial environment. Based on their presence in extant embryophytes and closely-related green algae, the first plants to survive on land likely possessed genes encoding channels with homology to large-conductance calcium-activated K+ channels (BK channels from the Slo family) in addition to primary voltage-gated potassium channels from the plant VG-type family (Shaker or Kv channels). While the function and gating of Shaker channels has been characterised in flowering plants, so far knowledge of BK channels has been limited to animal models. In humans, BK-mediated K+ efflux has a critical role in sperm motility and membrane polarisation to enable fertilisation. In the liverwort Marchantia polymorpha, the MpBK2a channel gene is most highly expressed in male reproductive tissue, suggesting that these channels may function in sexual reproduction. We characterised MpBK2a channels and found them to be strongly K+-selective, outward-rectifying, 80-pS channels capable of repolarising the membrane after stimulus-dependent depolarisation. In contrast to its animal counterpart, MpBK2a is insensitive to cytoplasmic Ca2+ variations but effectively gated by pH changes. Given that this plant BK channel is active even in the presence of trace amounts of external K+ and at low pH, the liverwort channel could have stabilised the membrane potential under stressful pre-historic conditions including nutrient-depleted and acid environments as early plant pioneers conquered land.

plant biology↗

bioRxiv2021

Regulation of large-conductance Ca2+- and voltage-gated K+ channels by electrostatic interactions with auxiliary β subunits.

Large-conductance Ca2+- and voltage-gated K+ (BK KCa1.1) channel complexes include pore-forming Slo1 subunits and often auxiliary {beta} subunits, latter of which noticeably modify the channels pharmacological and gating characteristics. In the absence of intracellular Ca2+, {beta}1 and {beta}4 modestly shift the overall voltage dependence of the channel to the positive direction by decreasing the probability that the ion conduction gate is open without any allosteric influence from the channels voltage or Ca2+ sensors. This intrinsic open probability is also critically regulated by the intracellular-facing 329RKK331 segment of human Slo1 (hSlo1) downstream of the transmembrane segment S6 in association with two negatively charged residues in S6 (E321 and E324) (Tian et al., Proc Natl Acad Sci USA, 116, 8591-8596, 2019). This study examined how {beta}1/{beta}4 and the RKK segment function together to control the channel gate. With select mutations in the RKK segment, inclusions of {beta}1 or {beta}4 can dramatically increase the intrinsic gate opening probability and shift the overall voltage dependence of the channel to the negative direction by up to 200 mV without Ca2+. This remarkable shift is mediated at least in part by electrostatic interactions between the Slo1 RKK and {beta} N-terminal segments as suggested by the results of double-mutant cycle analysis, ionic strength experiments, and molecular modelling. With or without auxiliary {beta} subunits, the Slo1 RKK and E321/E324 segments are thus critical determinants of the intrinsic open probability of the ion conduction gate and changes in the electrostatic environment near the RKK-EE segments are a potential mechanism of pharmacological gating modifiers.

physiology↗