Journal of General Physiology

TRPA1 (transient receptor potential ankyrin 1) is a non-selective, calcium-permeable cation channel that mediates pain by detecting environmental irritants and thermal stimuli. Although the role of TRPA1 in modulating pain perception is relatively well established, so far only a few human TRPA1 variants (N855S and A172V) have been associated with inherited neuropathic pain disorders. Here, we describe a novel TRPA1 variant (p. M978V) identified in two human subjects presenting with painful sensory neuropathy. Electrophysiological recordings demonstrate that the M978V variant confers gain-of-function properties to the TRPA1 channel, especially in response to allyl isothiocyanate (AITC; mustard oil), a well-characterised TRPA1 agonist. The M978V substitution enhances current density and shifts the half-maximal activation potential, rendering the channel more readily activated by electrophilic agonists, such as AITC. Furthermore, the mutant channel exhibits increased plasma membrane expression following AITC stimulation, suggesting that this single amino acid substitution affects both channel gating and trafficking. Using all-atom molecular dynamics simulation (MD), we highlighted that the variant is adjacent to the PIP2 binding site on the TRPA1 channel. We further show that depletion of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) increases current density in both WT and M978V channels. Importantly, the gain-of-function phenotype conferred by the M978V variant in response to AITC is dependent on the presence of PIP2. Collectively, our findings provide further evidence supporting the role of TRPA1 in human painful channelopathies and identify a previously unrecognised PIP2-dependent mechanism that regulates TRPA1 gain-of-function. Significance StatementIn this study we characterised the mechanism by which a rare TRPA1 variant leads to painful sensory neuropathy and discovered a novel modulatory PIP2-mediated regulation. Our in vitro data show that the variant confers gain-of-function properties to TRPA1 by enhancing its current density and open probability, as well as the channels surface membrane expression, in response to AITC, a known TRPA1 agonist. We also identified a novel interaction site for PIP2, a modulatory anionic lipid in the membrane of TRP channels. We have shown that abolishing endogenous PIP2 facilitates TRPA1 channel activation and that PIP2 is necessary for the variants gain-of-function properties, highlighting a new potential therapeutic avenue for neuropathic pain disorders.

Complete loss of function of the voltage-gated sodium channel subtype Nav1.7, encoded by SCN9A, results in congenital insensitivity to pain. Here, we investigate a previously identified variant, M899I, in which methionine at position 899 is substituted by isoleucine. This variant was originally described in a Chinese patient with loss of pain. We confirmed membrane expression of the mutant channel in HEK cells using extracellular HA-tagging; however, no sodium currents were detectable from the variant in patch-clamp recordings. The M899I substitution is located within a tightly packed hydrophobic region of the pore module. Introducing the corresponding variant into Nav1.2 and Nav1.5 similarly abolished channel function, underscoring the high conservation and functional importance of this residue. To further investigate the underlying mechanism, we combined in-silico coarse-grained molecular dynamics simulations with in-vitro electrophysiological analysis. Our simulations predicted that the M899I substitution induces collapse of the outer pore, substantially reducing both pore radius and volume. Substitution with other hydrophobic residues was likewise predicted to alter pore geometry and, consequently, ion permeation to varying degrees. Whole-cell voltage-clamp recordings validated these predictions, with observed current densities closely correlating with the extent of pore collapse predicted in silico. Together, our findings establish pore collapse as a mechanism underlying disease-relevant loss-of-function variants in Nav1.7 and suggest that this principle may extend to other sodium channel subtypes. Moreover, our results demonstrate that in-silico molecular dynamics approaches can reliably predict structural and functional consequences of channel mutations, as confirmed by in-vitro electrophysiological data.

The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2+ or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.

Myosin II generates force through the collective action of mechanically coupled motor ensembles, yet the mechanisms by which these ensembles sense changes in motor occupancy and coordinate force generation remain poorly understood. Ensemble force production may be governed by an optimal balance between effective motor occupancy and mechanical coordination rather than by motor number alone. We reconstituted cardiac myosin ensembles and systematically perturbed effective motor occupancy using the small-molecule drugs omecamtiv mecarbil (OM), which prolongs actomyosin interactions, and mavacamten (MAVA), which reduces the number of available force-generating myosin heads. Optical trapping measurements of full-length and S1 cardiac myosin ensembles revealed that force generation depended on both myosin concentration and pharmacological perturbation. Reducing myosin concentration increased force generation in the absence of drug, while OM and MAVA produced responses that varied with the initial occupancy state of the ensemble. Low concentrations of MAVA enhanced force generation under high motor occupancy but reduced force under low motor occupancy, whereas OM produced occupancy-dependent changes in both endpoint force and force dynamics. Force traces further revealed changes in the persistence and temporal coordination of force generation. These findings support a model in which cardiac myosin ensembles operate along an occupancy-coordination landscape, where maximal force generation is achieved at an intermediate level of effective motor occupancy. Our results illuminate how changes in motor occupancy are translated into coordinated ensemble mechanics and suggest that emergent mechanical feedback through the shared actin filament may enable ensembles to collectively sense and adapt to their mechanical state. Significance StatementForce generation by muscle emerges from the coordinated activity of myosin ensembles, yet the principles governing this collective behavior remain poorly understood. Using an in vitro force assay with full-length and truncated cardiac myosin, we systematically perturbed ensemble activity by varying myosin availability and pharmacologically altering the fraction of force-generating motors. We find that force production depends on an optimal balance of motor engagement rather than a simple increase or decrease in active motors, demonstrating that collective mechanical output arises from coordinated interactions within the ensemble. These findings reveal emergent design principles that govern molecular motor function and establish effective motor occupancy as a key regulator of collective force generation, providing new insight into the mechanisms underlying muscle contractility.

Collective cell migration is a fundamental process driving tissue repair, angiogenesis, and vascular homeostasis. This coordinated movement requires both intercellular communication via gap junctions and precise intracellular Ca{superscript 2} signaling, largely regulated by the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump within the endoplasmic reticulum (ER). Historically, carbenoxolone (CBX)--a synthetic derivative of glycyrrhetinic acid--has been widely utilized as a pharmacological tool to inhibit gap junctions and dissect their role in collective cell motility. However, its molecular specificity remains highly controversial. In the present study, using different cellular models, we found that CBX drastically reduces collective cell migration by a previously undescribed function for CBX: a fast, potent, and reversible inhibition of the SERCA pump, which provokes a passive leak of the luminal ER Ca{superscript 2} store. Our findings suggest that the effect of CBX over many cellular responses including cell migration and communication, previously only attributed to gap junction blockade, are indeed the consequence of the disruption of intracellular Ca{superscript 2} homeostasis. One Sentence Summarycarbenoxolone blocks cell migration by inhibiting SERCA

The {beta}-cardiac myosin (MYH7) mutation E525K was first identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered myosin constructs has shown that this mutation causes hypocontractility by stabilizing the interacting heads motif (IHM) of myosin despite the mutant E525K motor head exhibiting increased ATPase activity. However, no measurements have been made in myofilaments or cardiomyocytes to determine how this mutation affects contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell (iPSC)-derived cardiomyocytes engineered for heterozygous expression of E525K. Contraction of E525K single cells decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal isometric force in isolated myofibrils increased by 45%. Structural analysis revealed reduced myofibril content (13.7% decrease) and organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, no change was observed in the rate of ADP release. Importantly, there was no change in the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that myosin crossbridge cycling is not altered under load by E525K. Decreased force generation in EHTs and shortening in cardiomyocytes arise from reduced sarcomere number and myofibrillar disorganization. Additional force deficits likely result from stabilization of the IHM, as recently reported by others. This study demonstrates the value of multi-scale analysis for determining the functional profile of cardiomyocytes containing disease-related sarcomere protein mutations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@5f85f5org.highwire.dtl.DTLVardef@153b3b6org.highwire.dtl.DTLVardef@3b8f21org.highwire.dtl.DTLVardef@31d323_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract: A Model for how the E525K mutation impacts contracting myofibrils Here we have shown that the E525K mutation impacts contraction in three ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability. (4) The rate limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation and the rate of loaded contraction and relaxation is unchanged at all scales of contraction measured here. C_FIG

Arginine vasopressin (AVP) increases excitability of myometrial smooth muscle cells (MSMCs) through Gq-coupled AVP receptors. Although excitability requires membrane depolarization, the mechanisms linking AVP receptor activation to membrane depolarization and Ca{superscript 2} signaling are incompletely understood. Here, we show that AVPR1 is the predominant AVP receptor in primary MSMCs. In Xenopus oocytes, AVP signals through AVPR1 to inhibit SLO2.1-mediated potassium currents, reducing current amplitude to approximately 60% of control currents. Consistent with suppression of a hyperpolarizing conductance, AVP depolarized a myometrial cell line (hTERT-HM) and increased intracellular Ca{superscript 2} signaling. Analysis of Ca{superscript 2} dynamics revealed that the initial Ca{superscript 2} peak was largely preserved under conditions limiting extracellular Ca{superscript 2} entry, consistent with intracellular store release. Conversely, the oscillatory phase depended on extracellular Ca{superscript 2} influx and was reduced by SLO2.1 knockdown. Together, these findings support a model in which AVP preferentially signals through AVPR1A to inhibit SLO2.1, depolarize myometrial cells, enhance VDCC-dependent Ca{superscript 2} entry, and promote excitability, enhancing conditions for uterine contraction.

Reaction diffusion (RD) systems play a fundamental role in numerous biochemical and biophysical processes. Here, we present a novel analytical framework for solving RD equations by applying the Wentzel Kramers Brillouin Jeffreys (WKBJ) formalism to Ca nanodomains generated by individual membrane channels, a widely used paradigm for intracellular Ca signaling. Previous models have primarily focused on stationary Ca nanodomains while neglecting diffusion and saturation of intracellular Ca buffers and sensors. In contrast, we derive analytical solutions without these simplifying assumptions. Our analysis demonstrates that sustained Ca influx generates continuously expanding distributions of free Ca, whereas Ca bound buffers and sensors propagate as traveling waves. These predictions are supported experimentally by measurements of one-dimensional fluorescence profiles produced by single-channel activity and two-dimensional profiles generated by whole cell Ca currents. The analytical framework developed here readily extends Michaelis Menten type kinetics to reaction diffusion systems and may therefore be broadly applicable to biochemical and biophysical processes in which diffusion cannot be neglected.

Environmental temperature strongly influences muscle contractile mechanics and locomotor performance in ectotherms, yet animals routinely develop across a range of temperatures while maintaining effective movement. We tested the hypothesis that developmental temperature induces compensatory changes in the intrinsic mechanical properties of the muscles that power the fast-start escape response in larval zebrafish (Danio rerio). Larvae were reared at 25{degrees}C, 28{degrees}C, or 32{degrees}C, and contractile properties of intact tail myotomal muscles were measured across experimental temperatures. Acute changes in experimental temperature strongly affected twitch kinetics, particularly relaxation rate (Q10 = 2.1), resulting in substantial changes in twitch duration. In contrast, rearing temperature produced adaptive changes that opposed these acute thermal effects. At a common experimental temperature, muscles from cold-reared larvae exhibited faster intrinsic relaxation and greater force production during shortening at a physiologically relevant velocity, whereas warm-reared larvae showed slower relaxation and reduced shortening force. As a result, twitch kinetics were largely normalized when measurements were made at each group's rearing temperature, reducing the apparent thermal sensitivity of relaxation rate (Q10 = 1.1). To identify molecular correlates of these functional adaptations, we performed label-free quantitative LCMS proteomic analysis. Cold rearing increased the abundance of Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) proteins, driven primarily by elevated atp2a1 expression, while warm rearing reduced the abundance of the major parvalbumin isoforms pvalb1 and pvalb2. These changes implicate remodeling of intracellular calcium handling as a mechanism underlying thermal compensation of muscle function. Together, our results demonstrate that developmental temperature modifies the intrinsic mechanical properties of larval zebrafish muscle in ways that counteract the direct effects of environmental temperature, thereby preserving the timing and power-generating capacity required for fast-start escape performance.

RationaleSCN1B encodes the {beta}-subunits of the main cardiac voltage-gated sodium channel, NaV1.5. Variants are linked to cardiac conduction disease, often with concealed phenotypes. Whether {beta}1-subunits regulate conduction through nanoscale intercalated disc (ID) structures, e.g. perinexi, and ephaptic coupling remains unresolved. ObjectiveTest whether Scn1b haploinsufficiency induces latent conduction abnormalities that are unmasked by perturbations in extracellular nanodomains. Methods and ResultsAdult Scn1b+/- mice and wild-type (WT) littermates underwent multiscale phenotyping (qRT-PCR, Western blot, patch clamp, transmission electron microscopy (TEM), ex vivo optical mapping, in vivo ECG). Scn1b+/- hearts showed [~]50% reductions in Scn1b mRNA and {beta}1 protein without changes in canonical conduction proteins. Peak sodium current, baseline conduction velocity ex vivo, and baseline QRS duration in vivo were unchanged. However, TEM revealed increased baseline perinexal width in Scn1b+/- hearts. Osmotic expansion of the perinexus with mannitol slowed conduction to a greater extent in Scn1b+/- hearts and prolonged QRS duration in vivo. In contrast, perinexal narrowing with dextran 2MDa selectively increased conduction velocity in Scn1b+/- hearts. ConclusionsScn1b haploinsufficiency preserves baseline excitability and conduction but structurally remodels the ID at the nanoscale, increasing sensitivity to extracellular nanodomain perturbations. These data support a structural role for {beta}1-subunits in ephaptic coupling, and that conduction is maintained over a range of perinexal widths with pathological conduction slowing occurring beyond a critical width. Importantly, osmotic stress unmasks a concealed conduction phenotype, identifying extracellular nanodomain stability as a potential therapeutic target to mitigate arrhythmia risk in SCN1B-associated disease.

Length and time constants are foundational to the study of conduction in neurons and other biological cables but are exactly defined only for passive membranes. Here we define and derive exact length and time constants for propagating action potentials in unmyelinated axons. This derivation exploits specific instants during action potential conduction when the net transmembrane ionic current is zero, but axial current remains non-zero. At these instants, we define a curvature parameter,{kappa} , explore its determinants using computer modelling, demonstrate that it is the local real Laplace exponent of the action potential upstroke, and suggest practical approaches for its experimental measurement. From{kappa} , we define action potential length and time constants, {lambda}AP = 1/{surd}({kappa}racm) and {tau}AP = 1/{kappa}, and show that action potential propagation velocity is exactly {lambda}AP/{tau}AP.

Cav1.2 is an L-type voltage-gated Ca2+ channel (VGCC) that supports Ca2+ influx in response to membrane depolarization. Ca2+ entering via Cav1.2 alters gene expression, activates Ca2+-dependent enzymes and has been implicated in synaptic plasticity. ORL-1 is a Gi/o-coupled G protein-coupled receptor (GPCR) that is expressed in the peripheral and central nervous systems. Both Cav1.2 and ORL-1 are expressed in the hippocampus, where they have been implicated in learning and memory. It is well-documented that ORL-1 interacts with another VGCC, Cav2.2. However, less is known about potential interactions between Cav1.2 and ORL-1. Here, we examine the interplay between Cav1.2 (Cav1c, Cav2{delta}-1, Cav{beta}1) and ORL-1 co-expressed in tsA-201 cells by using biochemical, electrophysiological and confocal imaging analysis. Co-immunoprecipitations revealed that ORL-1 independently interacts with Cav1c and Cav2{delta}-1 subunits of the Cav1.2 channel complex. Electrophysiological recordings revealed that co-expression with ORL-1 reduced Cav1.2 peak current density without altering its biophysical properties. Acute perfusion with the ORL-1 receptor agonist nociceptin (1 M) did not alter Cav1.2 current density. Confocal imaging experiments revealed that ORL-1 significantly decreases Cav1.2 plasma membrane expression by disrupting forward trafficking. Interestingly, ORL-1 did not affect Cav1.2 endocytosis. Overall, our results demonstrate a previously unrecognized interaction between ORL-1 and Cav1.2 that alters Cav1.2 membrane expression without affecting biophysical properties.

Age-related reductions in muscle fiber size and contractile function, particularly in fibers expressing fast myosin heavy chains, contribute to declines in whole-muscle power. However, methodological limitations in estimating fiber size during contractile experiments have likely contributed to conflicting findings regarding whether reduced single-fiber force and power in older adults reflects their smaller size and/or impaired intrinsic contractile function. To address this, we coupled single-fiber contractile experiments with 3D-imaging in 7 young (19-40yrs) and 6 older (69-84yrs) males to assess intrinsic contractile function and compare agreement between 3D-derived cross-sectional area (CSA) and CSA estimates obtained either in air or solution. Fast fiber CSA from older males were [~]28-45% smaller across measurement conditions compared with young, whereas slow fiber CSA did not differ. Accordingly, absolute force and power of fast fibers were 41% and 37% lower. When normalized to CSA from measurements in air or 3D-imaging, size-specific force and power either did not differ or were greater in older adults, indicating preserved intrinsic contractile function in both fiber types. This was supported by no age-related differences in the rate of tension redevelopment (ktr), a size-independent measure of intrinsic contractile function. In contrast, size-specific force and power calculated using solution-based CSA estimates were lower in older compared with young adults, and Bland-Altman analyses demonstrated the poorest agreement between solution-based and 3D CSA measurements. These findings indicate that intrinsic contractile function is preserved with aging and suggest that methodological differences in CSA measurement contributes to the disparate findings in the literature.

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

Predictions of skeletal muscle energy consumption under a diverse range of muscle contractile conditions are critical for improving our understanding of locomotion. Existing mathematical models, while capturing the mechanical dependence of energy consuming processes, neglect the time-dependent behaviour and recovery costs associated with regenerating ATP. This time-dependence is important for predicting the energetic response of muscles during repetitive or cyclical tasks like locomotion, where muscle undergoes many contraction cycles. This study presents a novel model to predict energetic rates based on physiological processes: Ca2+ transport costs, cross-bridge cycling costs, and ATP regeneration. Previous mathematical models include the dependence on Ca2+ transport and cross-bridge cycling, but neglect the time-dependent response and the subsequent recovery of ATP following the contraction. Model parameters were obtained from existing data on isolated muscle preparations, and predicted energetic rates were validated on separate datasets across a range of contractile conditions including dynamic, sub-maximal, and twitch contractions. The time-dependent model was able to capture the influence of contraction frequency on peak energetic rates and the time-course of energetic recovery observed experimentally. The model captures key physiological processes while maintaining a minimal number of free parameters and low computational cost. This enables generalisability across muscles and species, and implementation into larger scale musculoskeletal models.

G protein-gated inwardly rectifying potassium (GIRK2) channels regulate neuronal excitability and are implicated in neurodevelopmental disorders. A rare KCNJ6 variant, G154C (hGIRK2G154C), was identified in a patient with mild Keppen-Lubinsky syndrome features, contrasting with severe phenotypes linked to other selectivity filter mutations. Here we combined molecular dynamics simulations and patch-clamp electrophysiology to characterize the hGIRK2G154C mutant, revealing a widened selectivity filter that resulted in loss of potassium selectivity, aberrant sodium permeation, and loss of inward rectification, indicating a severe gain-of-function phenotype. An in silico and electrophysiological drug screen identified FDA-approved compounds, including nefazodone and eletriptan, that potently inhibited GIRK2 and GIRK2G154C through distinct blocking mechanisms. These findings elucidate the structural and functional impact of the G154C mutation and highlight potential pharmacological tools and therapeutic candidates for the treatment of GIRK2 channelopathies.

Gain-of-function (GoF) variants in the Kv7.2 channel are associated with a clinically relevant subset of neurodevelopmental disorders. While most GoF substitutions identified so far affect the voltage-sensing domain, we recently described three mutations in the intracellular-facing activation gate (AG), G313S, A317T, and L318V. Electrophysiological recordings showed that these variants increase macroscopic current density and enhance channel open probability. Consistently, molecular dynamics (MD) simulations revealed that they hinder complete channel closure by destabilizing the closed AG and increasing hydration of the central cavity (CC). Whether this partially open conformation can support K+ permeation, however, remained an open question. Here, we combined long-timescale atomistic simulations and simulations with applied electric fields to evaluate the stability of these mutant-associated AG states over longer timescales and their functional relevance. In new trajectories, all three substitutions consistently shifted the closed intracellular gate toward a widened, water-accessible conformation, accompanied by increased CC hydration. We then assessed the functional significance of this partially open state by simulating the A317T channel under applied electric fields. The conformation supported K+ translocation, whereas the closed WT pore remained impermeable under all tested voltages. When simulations were started from open channel conformations, both WT and A317T conducted K+ ions, indicating that the main effect of the substitution is to destabilize closure of the intracellular gate rather than to alter the fully conductive open state. Together, these data show that AG GoF variants can generate an intermediate gate conformation that permits ion permeation, providing a mechanistic link between mutant-induced pore remodeling and Kv7.2 dysfunction in KCNQ2-related disease.

AbstractPhospholipid asymmetry is a hallmark of mammalian cell membranes and reflects the selective distribution of distinct phospholipid species between the two leaflets of the lipid bilayer. Although this asymmetry is tightly maintained, the membrane proteins whose functions depend on it remain largely unknown. To perturb phospholipid asymmetry experimentally, we expressed a constitutively active phospholipid scramblase and thereby identified transient receptor potential melastatin 8 (TRPM8) as an ion channel regulated by this membrane property. Activation of TRPM8 by both l-menthol and innocuous cold was markedly suppressed following disruption of phospholipid asymmetry. Likewise, selective depletion of phosphatidylserine (PS), a phospholipid enriched in the cytoplasmic leaflet, using a cytosolically targeted PS decarboxylase attenuated TRPM8 activation, indicating that cytoplasmic PS is required for proper TRPM8 function. Mechanistically, our findings suggest that cytoplasmic PS supports efficient TRPM8 activation by maintaining the biochemical state of the channel. Together, these findings identify TRPM8 as a phospholipid asymmetry-dependent ion channel and establish an experimental strategy for systematically identifying membrane proteins regulated by phospholipid asymmetry. This work provides a foundation for future studies investigating the biological significance of this fundamental membrane property.

Every known life form senses and reacts to mechanical forces. These mechanical stimuli can be converted into electrical signals by mechanically gated ion channels, a transduction cascade pivotal to numerous physiological functions including touch, hearing, mechanical pain, circulation, gastrointestinal function, and mechanical loading in various tissues. Despite continuous efforts, numerous mechanically gated ion channels with the mechanotransduction process underlying these physiological functions remain unidentified. Here, we focused on the transmembrane channel-like (TMC) protein family expressed in the cultured cells to identify those with potential mechanosensitive activity. Remarkably, in contrast to human TMC1/2 (HsTMC1/2), human TMC3-8 (HsTMC3-8) proteins are localized to the plasma membrane when heterologously expressed in the cultured cells. Further experiments revealed that mechanical poking stimuli can effectively activate HsTMC3-8. In addition, HsTMC3-8 induced stretch-activated currents and elicited well-resolved single-channel activities in response to negative pressure stimulation. The mutants near the putative pore region altered reversal potentials (Erev) of HsTMC3-8, suggesting that TMC3-8 are likely pore-forming subunits of ion channels. In summary, we proposed that TMC proteins are the largest mammalian mechanically gated ion channel family.

Intracellular calcium waves in cardiac myocytes propagate through a fire-diffuse-fire mechanism in which calcium released from one RyR2 cluster diffuses to neighboring clusters and triggers their activation. Yet propagation faces a fundamental physical difficulty: the calcium signal must cross distances of 1-2 {micro}m between Z-planes while being attenuated by cytosolic buffering and diffusion, and at the same time the release site depletes its local sarcoplasmic reticulum calcium store. How waves propagate efficiently despite these constraints has remained unclear. We developed a three-dimensional computational model of mouse ventricular myocytes at 100 nm resolution to address this question. Our central finding is that co-localization of calsequestrin2 (CASQ2) with RyR2 clusters is required for robust wave propagation. In a physiological model, where CASQ2 is concentrated at release sites as observed experimentally, calcium waves propagate reliably across the cell with velocities that match the experimental range. In contrast, when CASQ2 is distributed uniformly throughout the sarcoplasmic reticulum, keeping total CASQ2 unchanged, the wavefront stalls. These results identify CASQ2-RyR2 co-localization as a key structural requirement for effective calcium wave propagation in ventricular myocytes. Author summaryCalcium waves in cardiomyocytes are thought to underlie the onset of malignant cardiac arrhythmias, such as ventricular tachycardia and fibrillation. Yet, the specific conditions that regulate the transition from local calcium sparks to sustained waves remain poorly understood. Using a newly developed computational model of calcium handling, we demonstrate that the spatial distribution of key regulatory proteins is a critical determinant of arrhythmogenicity. Specifically, we found that calsequestrin2, which buffers Ca2+ within the sarcoplasmic reticulum, must be strictly colocalized with Ca2+ release proteins to facilitate sustained wave propagation. This discovery suggests that cardiac stability depends less on the total quantity of protein and more on its precise architectural organization. The consequences of this finding are significant: it implies that "spatial dysregulation"--where proteins are present but mislocalized--may be a hidden driver of arrhythmias even when protein levels appear normal. This shifts the therapeutic focus from simply altering ion channel conductance to preserving or restoring the structural tethering of the junctional SR. By focusing on the nanodomain architecture, we can better understand how cellular remodeling leads to life-threatening electrical instability.