Journal of General Physiology最新文献

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.

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.

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.

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.

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.

In a new JGP study (Thoreson et al. https://doi.org/10.1085/jgp.202413746), anatomically realistic simulations reveal how the complex architecture of rod synapses influences glutamate dynamics and postsynaptic responses.

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.

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.

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.