Journal of General Physiology (JGP)

Studies of potassium channel evolution from the Jegla group contribute valuable insights into the evolution of complexity in electrical signaling and the conservation and repurposing of key molecular components throughout evolutionary history.

The ctenophore species Mnemiopsis leidyi is known to have a large set of voltage-gated K⁺ channels, but little is known about the functional diversity of these channels or their evolutionary history in other ctenophore species. Here, we searched the genomes of two additional ctenophore species, Beroe ovata and Hormiphora californensis, for voltage-gated K⁺ channels and functionally expressed a subset of M. leidyi channels. We found that the last common ancestor of these three disparate ctenophore lineages probably had at least 33 voltage-gated K⁺ channels. Two of these genes belong to the EAG family, and the remaining 31 belong to the Shaker family and form a single clade within the animal/choanoflagellate Shaker phylogeny. We additionally found evidence for 10 of these Shaker channels in a transcriptome of the early branching ctenophore lineage Euplokamis dunlapae, suggesting that the diversification of these channels was already underway early in ctenophore evolution. We functionally expressed 16 Mnemiopsis Shakers and found that they encode a diverse array of voltage-gated K⁺ conductances with functional orthologs for many classic Shaker family subtypes found in cnidarians and bilaterians. Analysis of Mnemiopsis transcriptome data show these 16 Shaker channels are expressed in a wide variety of cell types, including neurons, muscle, comb cells, and colloblasts. Ctenophores therefore appear to have independently evolved much of the voltage-gated K⁺ channel diversity that is shared between cnidarians and bilaterians.

Marfan syndrome (MFS) is an autosomal dominant disease caused by mutations in the gene (FBN1) of fibrillin-1, a major determinant of the extracellular matrix (ECM). Functional impairment in the cardiac left ventricle (LV) of these patients is usually a consequence of aortic valve disease. However, LV passive stiffness may also be affected by chronic changes in mechanical load and ECM dysfunction. Passive stiffness is determined by the giant sarcomeric protein titin that has two main cardiac splice isoforms: the shorter and stiffer N2B and the longer and more compliant N2BA. Their ratio is thought to reflect myocardial response to pathologies. Whether this ratio and titin’s sarcomeric layout is altered in MFS is currently unknown. Here, we studied LV samples from MFS patients carrying FBN1 mutation, collected during aortic root replacement surgery. We found that the N2BA:N2B titin ratio was elevated, indicating a shift toward the more compliant isoform. However, there were no alterations in the total titin content compared with healthy humans based on literature data. Additionally, while the gross sarcomeric structure was unaltered, the M-band was more extended in the MFS sarcomere. We propose that the elevated N2BA:N2B titin ratio reflects a general adaptation mechanism to the increased volume overload resulting from the valvular disease and the direct ECM disturbances so as to reduce myocardial passive stiffness and maintain diastolic function in MFS.

The cholinergic interneurons (ChIs) of the nucleus accumbens (NAc) have a critical role in the activity of this region, specifically in the context of major depressive disorder. To understand the circuitry regulating this behavior, we sought to determine the areas that directly project to these interneurons by utilizing the monosynaptic cell-specific tracing technique. Mapping showed monosynaptic projections that are exclusive to NAc ChIs. To determine if some of these projections are altered in a depression mouse model, we used mice that do not express the calcium-binding protein p11 specifically in ChIs (ChAT-p11 cKO) and display a depressive-like phenotype. Our data demonstrated that while the overall projection areas remain similar between wild type and ChAT-p11 cKO mice, the number of projections from the ventral hippocampus (vHIP) is significantly reduced in the ChAT-p11 cKO mice. Furthermore, using optogenetics and electrophysiology we showed that glutamatergic projections from vHIP to NAc ChIs are severely altered in mutant mice. These results show that specific alterations in the circuitry of the accumbal ChIs could play an important role in the regulation of depressive-like behavior, reward-seeking behavior in addictions, or psychiatric symptoms in neurodegenerative diseases.

Integrating cellular sarcoplasmic reticulum (SR) Ca²⁺ release with the known Ca²⁺ activation properties of RyR2s remains challenging. The sharp increase in SR Ca²⁺ permeability above a threshold SR luminal [Ca²⁺] is not reflected in RyR2 kinetics from single-channel studies. Additionally, the current paradigm that global Ca²⁺ release (Ca²⁺ waves) arises from interacting local events (Ca²⁺ sparks) faces a key issue that these events rarely activate neighboring sites. We present a multiscale model that reproduces Ca²⁺ sparks and waves in skinned ventricular myocytes using experimentally validated RyR2 kinetics. The model spans spatial domains from 10⁻⁸ to 10⁻⁴ m and timescales from 10⁻⁶ to 10 s. Ca²⁺ release sites are distributed in cubic voxels (0.25-µm sides) informed by super-resolution micrographs. We use parallel computing to calculate Ca²⁺ transport, diffusion, and buffering. Substantial increases in SR Ca²⁺ release occur, and Ca²⁺ waves initiate when Ca²⁺ sparks become prolonged above a threshold SR [Ca²⁺]. These prolonged events (Ca²⁺ embers) are much more likely than Ca²⁺ sparks to activate release from neighboring sites and accumulate increases in cytoplasmic [Ca²⁺] along with an associated fall in Ca²⁺ buffering power. This primes the cytoplasm for Ca²⁺-induced Ca²⁺ release (CICR) that produces Ca²⁺ waves. Thus, Ca²⁺ ember formation and CICR are both essential for initiation and propagation of Ca²⁺ waves. Cell architecture, along with the differential effects of RyR2 opening and closing rates, collectively determines the SR [Ca²⁺] threshold for Ca²⁺ embers, waves, and the phenomenon of store overload–induced Ca²⁺ release.

Synapses of retinal rod photoreceptors involve deep invaginations occupied by second-order rod bipolar cell (RBP) and horizontal cell (HC) dendrites. Synaptic vesicles are released into this invagination at multiple sites beneath an elongated presynaptic ribbon. To study the impact of this architecture on glutamate diffusion and receptor activity, we reconstructed four rod terminals and their postsynaptic dendrites from serial electron micrographs of the mouse retina. We incorporated these structures into anatomically realistic Monte Carlo simulations of neurotransmitter diffusion and receptor activation. By comparing passive diffusion of glutamate in realistic structures with geometrically simplified models, we found that glutamate exits anatomically realistic synapses 10-fold more slowly than previously predicted. Constraining simulations with physiological data, we modeled activity of EAAT5 glutamate transporters in rods, AMPA receptors on HC dendrites, and metabotropic glutamate receptors (mGluR6) on RBP dendrites. Simulations suggested that ∼3,000 EAAT5 populate rod membranes. While uptake by surrounding glial Müller cells retrieves most glutamate released by rods, binding and uptake by EAAT5 influence RBP kinetics. Glutamate persistence allows mGluR6 on RBP dendrites to integrate the stream of vesicles released by rods in darkness. Glutamate’s tortuous diffusional path confers quantal variability, as release from nearby ribbon sites exerts larger effects on RBP and HC receptors than release from more distant sites. Temporal integration supports slower sustained release rates, but additional quantal variability can impede postsynaptic detection of changes in release produced by rod light responses. These results show an example of the profound impact that synaptic architecture can have on postsynaptic responses.

Two-pore channels (TPCs) are twofold symmetric endolysosomal cation channels forming important drug targets, especially for antiviral drugs. They are activated by calcium, ligand binding, and membrane voltage, and to date, they are the only ion channels shown to alter their ion selectivity depending on the type of bound ligand. However, despite their importance, ligand activation of TPCs and the molecular mechanisms underlying their ion selectivity are still poorly understood. Here, we set out to elucidate the mechanistic basis for the ion selectivity of human TPC2 (hTPC2) and the molecular mechanism of ligand-induced channel activation by the lipid PI(3,5)P2. We performed all-atom in silico electrophysiology simulations to study Na⁺ and Ca²⁺ permeation across full-length hTPC2 on the timescale of ion conduction and investigated the conformational changes induced by the presence or absence of bound PI(3,5)P2. Our findings reveal that hTPC2 adopts distinct conformations depending on the presence of PI(3,5)P2 and elucidate the allosteric transition pathways between these structures. Additionally, we examined the permeation mechanism, solvation states, and binding sites of ions during ion permeation through the pore. The results of our simulations explain the experimental observation that hTPC2 is more selective for Na⁺ over Ca²⁺ ions in the presence of PI(3,5)P2via a multilayer selectivity mechanism. Importantly, mutations in the selectivity filter region of hTPC2 maintain cation conduction but change the ion selectivity of hTPC2 drastically.

The article by Lewis et al. (https://doi.org/10.1085/jgp.202413709) in this issue of JGP describes the use of single-molecule fluorescence polarization microscopy to obtain estimates of all the rate constants for transitions in the catalytic cycle of AdiC, a bacterial transporter for arginine and agmatine that has been believed to be a 1:1 exchanger.

To understand the mechanism underlying the ability of individual AdiC molecules to transport arginine and agmatine, we used a recently developed high-resolution single-molecule fluorescence-polarization microscopy method to investigate conformation-specific changes in the emission polarization of a bifunctional fluorophore attached to an AdiC molecule. With this capability, we resolved AdiC’s four conformations characterized by distinct spatial orientations in the absence or presence of the two substrates, and furthermore, each conformation’s two energetic states, totaling 24 states. From the lifetimes of individual states and state-to-state transition probabilities, we determined 60 rate constants characterizing the transitions and 4 KD values characterizing the interactions of AdiC’s two sides with arginine and agmatine, quantitatively defining a 24-state model. This model satisfactorily predicts the observed Michaelis–Menten behaviors of AdiC. With the acquired temporal information and existing structural information, we illustrated how to build an experiment-based integrative 4D model to capture and exhibit the complex spatiotemporal mechanisms underlying facilitated transport of substrates. However, inconsistent with what is expected from the prevailing hypothesis that AdiC is a 1:1 exchanger, all observed conformations transitioned among themselves with or without the presence of substrates. To corroborate this unexpected finding, we performed radioactive flux assays and found that the results are also incompatible with the hypothesis. As a technical advance, we showed that a monofunctional and the standard bifunctional fluorophore labels report comparable spatial orientation information defined in a local frame of reference. Here, the successful determination of the complex conformation-kinetic mechanism of AdiC demonstrates the unprecedented resolving power of the present microscopy method.

JGP study (Avilés et al. https://doi.org/10.1085/jgp.202413642) reveals that visual perception of high-frequency flickers requires signaling by the tissue polarity protein FAT3 in retinal bipolar cells.

α-actinin (ACTN) is a pivotal member of the actin-binding protein family, crucial for the anchoring and organization of actin filaments within the cytoskeleton. Four isoforms of α-actinin exist: two non-muscle isoforms (ACTN1 and ACTN4) primarily associated with actin stress fibers and focal adhesions, and two muscle-specific isoforms (ACTN2 and ACTN3) localized to the Z-disk of the striated muscle. Although these isoforms share structural similarities, they exhibit distinct functional characteristics that reflect their specialized roles in various tissues. Genetic variants in α-actinin isoforms have been implicated in a range of pathologies, including cardiomyopathies, thrombocytopenia, and non-cardiovascular diseases, such as nephropathy. However, the precise impact of these genetic variants on the α-actinin structure and their contribution to disease pathogenesis remains poorly understood. This review provides a comprehensive overview of the structural and functional attributes of the four α-actinin isoforms, emphasizing their roles in actin crosslinking and sarcomere stabilization. Furthermore, we present detailed structural modeling of select ACTN1 and ACTN2 variants to elucidate mechanisms underlying disease pathogenesis, with a particular focus on macrothrombocytopenia and hypertrophic cardiomyopathy. By advancing our understanding of α-actinin’s role in both normal cellular function and disease states, this review lays the groundwork for future research and the development of targeted therapeutic interventions.

Gupta et al. (https://doi.org/10.1085/jgp.202413676) reconcile a disconnect between structural and functional data regarding stoichiometry of PANX1 channels and provide new insights about channel activation.

Vision is initiated by the reception of light by photoreceptors and subsequent processing via downstream retinal neurons. Proper circuit organization depends on the multifunctional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here, we investigated the retinal function of Fat3 mutant mice and found decreases in both electroretinography and perceptual responses to high-frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. The role of FAT3 in the response to high temporal frequency flashes depends upon its ability to transduce an intracellular signal. Mechanistically, FAT3 binds to the synaptic protein PTPσ intracellularly and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. These findings expand the repertoire of FAT3’s functions and reveal its importance in bipolar cells for high-frequency light response.

Patients with periodic paralysis have attacks of weakness precipitated by depolarization of muscle. Each form of periodic paralysis is associated with unique changes in serum K⁺ during attacks of weakness. In hypokalemic periodic paralysis (hypoKPP), the mutation-induced gating pore current causes weakness associated with low serum K⁺. In hyperkalemic periodic paralysis (hyperKPP), mutations increase a non-inactivating Na⁺ current (Na persistent or NaP), which causes weakness associated with elevation of extracellular K⁺. In Andersen–Tawil syndrome, mutations causing loss of Kir channel function cause weakness associated with either low or high K⁺. We developed a computer model to address two questions: (1) What mechanisms are responsible for the distinct K⁺ dependencies of muscle depolarization-induced weakness in the three forms of periodic paralysis? (2) Why does extracellular K⁺ become elevated during attacks of weakness in hyperKPP, reduced in hypoKPP, and both elevated and reduced in Andersen–Tawil syndrome? We experimentally tested the model assumptions about resting potential in normal K⁺ solution in hyperKPP and hypoKPP. Recreating the distinct K⁺ dependence of all three forms of periodic paralysis required including the K⁺ and voltage dependence of current through Kir channels, the extracellular K⁺ and intracellular Na⁺ dependence of the Na/K ATPase activity, and the distinct voltage dependencies of the gating pore current and NaP. A key factor determining whether muscle would depolarize was the direction of small net K⁺ and net Na⁺ fluxes, which altered ion concentrations over hours. Our findings may aid in development of novel therapy for diseases with dysregulation of muscle excitability.

Phan and Fitzsimons (https://doi.org/10.1085/jgp.202413582) develop a new mathematical model of muscle contraction that explores cooperative mechanisms in small (murine) and large (porcine) myocardium.

Voltage-gated Ca²⁺ channels (VGCCs) are highly expressed throughout numerous biological systems and play critical roles in synaptic transmission, cardiac excitation, and muscle contraction. To perform these various functions, VGCCs are highly regulated. Inactivation comprises a critical mechanism controlling the entry of Ca²⁺ through these channels and constitutes an important means to regulate cellular excitability, shape action potentials, control intracellular Ca²⁺ levels, and contribute to long-term potentiation and depression. For CaV1 and CaV2 channel families, inactivation proceeds via two distinct processes. Voltage-dependent inactivation (VDI) reduces Ca²⁺ entry through the channel in response to sustained or repetitive depolarization, while Ca²⁺-dependent inactivation (CDI) occurs in response to elevations in intracellular Ca²⁺ levels. These processes are critical for physiological function and undergo exquisite fine-tuning through multiple mechanisms. Here, we review known determinants and modulatory features of these two critical forms of channel regulation and their role in normal physiology and pathophysiology.

The mechanisms underlying cooperative activation and inactivation of myocardial force extend from local, near-neighbor interactions involving troponin-tropomyosin regulatory units (RU) and crossbridges (XB) to more global interactions across the sarcomere. To better understand these mechanisms in the hearts of small and large mammals, we undertook a simplified mathematical approach to assess the contribution of three types of near-neighbor cooperative interactions, i.e., RU-induced, RU-activation (RU–RU), crossbridge-induced, crossbridge-binding (XB–XB), and XB-induced, RU-activation (XB–RU). We measured the Ca²⁺ and activation dependence of the rate constant of force redevelopment in murine- and porcine-permeabilized ventricular myocardium. Mathematical modeling of these three near-neighbor interactions yielded nonlinear expressions for the RU–RU and XB–RU rate coefficients (kon and koff) and XB–XB rate coefficients describing the attachment of force-generating crossbridges (f and f’). The derivation of single cooperative coefficient parameters (u = RU–RU, w = XB–RU, and v = XB–XB) permitted an initial assessment of the strength of each near-neighbor interaction. The parameter sets describing the effects of discrete XB–XB or XB–RU interactions failed to adequately fit the in vitro contractility data in either murine or porcine myocardium. However, the Ca²⁺ dependence of ktr in murine and porcine ventricular myocardium was well fit by parameter sets incorporating the RU–RU cooperative interaction. Our results indicate that a significantly stronger RU–RU interaction is present in porcine ventricular myocardium compared with murine ventricular myocardium and that the relative strength of the near-neighbor RU–RU interaction contributes to species-specific myocardial contractile dynamics in small and large mammals.

Elhanafy et al. used Molecular Dynamics simulations and electrophysiology to show how identical mutations in the volgage sending domain of sodium channels can yield differential functional effects.

Voltage-gated sodium channels (VGSCs) in the peripheral nervous system shape action potentials (APs) and thereby support the detection of sensory stimuli. Most of the nine mammalian VGSC subtypes are expressed in nociceptors, but predominantly, three are linked to several human pain syndromes: while Nav1.7 is suggested to be a (sub-)threshold channel, Nav1.8 is thought to support the fast AP upstroke. Nav1.9, as it produces large persistent currents, is attributed a role in determining the resting membrane potential. We characterized the gating of Nav1.1–Nav1.3 and Nav1.5–Nav1.9 in manual patch clamp with a focus on the AP subthreshold depolarization phase. Nav1.9 exhibited the most hyperpolarized activation, while its fast inactivation resembled the depolarized inactivation of Nav1.8. For some VGSCs (e.g., Nav1.1 and Nav1.2), a positive correlation between ramp current and window current was detected. Using a modified Hodgkin–Huxley model that accounts for the time needed for inactivation to occur, we used the acquired data to simulate two nociceptive nerve fiber types (an Aδ- and a mechano-insensitive C-nociceptor) containing VGSC conductances according to published human RNAseq data. Our simulations suggest that Nav1.9 is supporting both the AP upstroke and its shoulder. A reduced threshold for AP generation was induced by enhancing Nav1.7 conductivity or shifting its activation to more hyperpolarized potentials, as observed in Nav1.7-related pain disorders. Here, we provide a comprehensive, comparative functional characterization of VGSCs relevant in nociception and describe their gating with Hodgkin–Huxley–like models, which can serve as a tool to study their specific contributions to AP shape and sodium channel–related diseases.

Voltage-gated sodium (Nav) channels are pivotal for cellular signaling, and mutations in Nav channels can lead to excitability disorders in cardiac, muscular, and neural tissues. A major cluster of pathological mutations localizes in the voltage-sensing domains (VSDs), resulting in either gain-of-function, loss-of-function effects, or both. However, the mechanism behind this functional diversity of mutations at equivalent positions remains elusive. Through hotspot analysis, we identified three gating charges (R1, R2, and R3) as major mutational hotspots in VSDs. The same amino acid substitutions at equivalent gating-charge positions in VSDI and VSDII of the cardiac sodium channel Nav1.5 show differential gating property impacts in electrophysiology measurements. We conducted molecular dynamics (MD) simulations on wild-type channels and six mutants to elucidate the structural basis of their differential impacts. Our 120-µs MD simulations with applied external electric fields captured VSD state transitions and revealed the differential structural dynamics between equivalent R-to-Q mutants. Notably, we observed transient leaky conformations in some mutants during structural transitions, offering a detailed structural explanation for gating-pore currents. Our salt-bridge network analysis uncovered VSD-specific and state-dependent interactions among gating charges, countercharges, and lipids. This detailed analysis revealed how mutations disrupt critical electrostatic interactions, thereby altering VSD permeability and modulating gating properties. By demonstrating the crucial importance of considering the specific structural context of each mutation, our study advances our understanding of structure–function relationships in Nav channels. Our work establishes a robust framework for future investigations into the molecular basis of ion channel–related disorders.

Small molecule inhibitors of the sodium channel are common pharmacological agents used to treat a variety of cardiac and nervous system pathologies. They act on the channel via binding within the pore to directly block the sodium conduction pathway and/or modulate the channel to favor a non-conductive state. Despite their abundant clinical use, we lack specific knowledge of their protein–drug interactions and the subtle variations between different compound structures. This study investigates the binding and accessibility of nine different compounds in the pore cavity of the Nav1.5 sodium channel using enhanced sampling simulations. We find that most compounds share a common location of pore binding—near the mouth of the DII–III fenestration—associated with the high number of aromatic residues in this region. In contrast, some other compounds prefer binding within the lateral fenestrations where they compete with lipids, rather than binding in the central cavity. Overall, our simulation results suggest that the drug binding within the pore is highly promiscuous, with most drugs having multiple low-affinity binding sites. Access to the pore interior via two out of four of the hydrophobic fenestrations is favorable for the majority of compounds. Our results indicate that the polyspecific and diffuse binding of inhibitors in the pore contributes to the varied nature of their inhibitory effects and can be exploited for future drug discovery and optimization.

Tao and Corry used metadynamics, an enhanced sampling method to identify and classify Nav channel blockers.

The physiological, functional, and structural properties of proteins and their pathogenic variants can be summarized using many tools. The information relating to a single protein is often spread among different sources requiring different programs for access. It is not always easy to select, simultaneously visualize, and compare specific properties of different proteins. On the other hand, comparing members of the same protein family could suggest conserved properties or highlight significant differences. We have thus developed a web interface, ALLIN (Annotation of sequence aLignment and structuraL proteIn visualizatioN) for the simultaneous visualization of multi-sequence protein alignments, including comments and annotations, and the related three-dimensional structures. This interface permits the inclusion of comments and coloring of residues in the alignment section, according to a user-defined color code, allowing a quick overview of specific properties. The interface does not require training or coding expertise, and the result is a unique “memo” web page that combines data from different sources, with the flexibility to highlight only the information of interest. The output provides an overview of the state of art of a protein family that is easily shared among researchers and new data can be conveniently added as it emerges. We believe the ALLIN tool can be useful for all scientists working on the structure–function analysis of proteins, in particular on those involved in human genetic diseases.