Journal of General Physiology

Journal of General Physiology (JGP)

Published by Rockefeller University Press

Online ISSN: 1540-7748

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Print ISSN: 0022-1295

Disciplines: Physiology, Membrane Protein Physiology, Protein Structure and Dynamics, Lipid and Membrane Biophysics, Cell Mechanics and Contractile Systems, Intracellular and Intercellular signaling

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Conductance-voltage or current-voltage relationship of activation and SSFI protocols. (A) Overview of all conductance-voltage curves derived from activation measurements. (B) Overview of all current-voltage curves derived from SSFI measurements (voltage protocol of SSFI depicted in inlet. Note that for Nav1.8 measurements, most cells were stimulated with a −110 to 0 mV voltage range). (C) Example current traces of Nav1.7 elicited by the activation protocol (voltage protocol depicted in inlet. Note that for Nav1.8 measurements, most cells were stimulated with a −60 to +70 mV voltage range). (D–J) Overlay of activation and inactivation curves for Nav1.1 (D), Nav1.2 (E), Nav1.3 (F), Nav1.5 (G), Nav1.6 (H), Nav1.7 (I), and Nav1.8 (J). Data are shown as mean ± SD and superimposed Boltzmann fit curve.
Activation and SSFI parameters and window current. (A) V 50 values obtained from activation Boltzmann fits. (B)V50 values obtained from SSFI Boltzmann fits. (C) AUC values underneath the superimposed activation and SSFI Boltzmann fit curves, i.e., the window current. Inlet: Schematic depiction of AUC calculation, x being the membrane voltage of the activation and SSFI Boltzmann fit curve intersection. (D) Slope values k obtained from activation Boltzmann fits. (E) Slope values k gathered from SSFI Boltzmann fits. (F) Bottom values gathered from SSFI Boltzmann fits, indicating the fraction of channels remaining open after complete SSFI. Data are shown as mean ± SD. Statistical significance from multiple comparisons has not been indicated in the figure panels for readability purposes but can be consulted in Tables S3, S4, S5, and S6.
of Na v 1.9 activation and SSFI data. Data from Leipold et al. (2013), Leipold et al. (2015) were pooled and analyzed concerning Nav1.9 gating and window currents. (A) Example current traces of Nav1.9 elicited by the activation protocol. Note that cells were stimulated with a −127 to + 63 mV voltage range. (B) Overlay of activation conductance-voltage and SSFI current-voltage curves. (C)V50 values obtained from activation and SSFI Boltzmann fits. (D) Slope values k obtained from activation and SSFI Boltzmann fits. (E) AUC values underneath the superimposed activation and SSFI Boltzmann fit curves, i.e., the window current. Data are shown as mean ± SD.
Example traces of Na v 1.7 ramp currents elicited by slowly depolarizing ramp stimuli. Voltage protocol depicted in inlet. Scale bars apply to current traces only.
Current-voltage parameters obtained from ramp current measurements. (A) Maximum inward current from ramp current measurement normalized to maximum inward current from activation measurements. (B) Voltage at which the maximum inward current occurred during ramp current measurement. Data are shown as box plots with whiskers indicating the 10th and 90th percentile and dots for measurements below the 10th and above the 90th percentile.

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Nociceptor sodium channels shape subthreshold phase, upstroke, and shoulder of action potentials

January 2025

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60 Reads

Phil Alexander Köster

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Journal of General Physiology (JGP) publishes mechanistic and quantitative cellular and molecular physiology of the highest quality. Est. 1918

Recent articles


FAT3 provides a flicker of light
  • Article

February 2025

Ben Short

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.


Structural and functional insights into α-actinin isoforms and their implications in cardiovascular disease
  • Literature Review
  • Publisher preview available

February 2025

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30 Reads

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.



ERG responses to high-frequency flickers require FAT3 signaling in mouse retinal bipolar cells

February 2025

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11 Reads

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1 Citation

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.


Mechanisms underlying the distinct K dependencies of periodic paralysis

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.




A hinged lid mechanism of VDI. (A) Cartoon depicting the hinged lid or door wedge mechanisms of VDI described for CaV1 channels. For the hinged lid, the I–II loop (blue) interacts with the distal S6 regions to occlude the channel. In the door wedge mechanism, the I–II loop inserts such that the channel activation gate is pushed closed. (B) Single-channel recordings of L-type channels within guinea pig ventricular myocytes with Ba²⁺ as the charge carrier. VDI is seen as a loss of channel openings toward the end of the trace (i.e., sweep 1 versus 6). Bottom: An average of many single-channel sweeps. Data reproduced from Yue et al. (1990) with permission from Science. (C) The β subunit (red) can modulate VDI through interaction with the I–II loop (blue), here shown for a hinged lid scenario. When β2a associates with the channel (left), the palmitoylation site within this subunit variant anchors β2a to the membrane (spring) inhibiting the I–II loop from occluding the pore. However, variants such as β1 lack this palmitoylation sequence and allow the I–II loop to move freely (right), permitting VDI.
An allosteric mechanism for CDI. (A) Cartoon depicting the known channel intracellular regions relevant to CaM-mediated CDI of CaV1 channels. (B) Cartoon of an allosteric model describing CDI. Channels initially open within the “mode 1” set of states. Upon Ca²⁺ entry and binding to CaM, channels transition to “Mode Ca,” which is characterized by a lower open probability resulting in CDI. Figure adapted from Dick et al. (2016). (C) Single-channel recordings of L-type channels within guinea pig ventricular myocytes with Ca²⁺ as the charge carrier. CDI is seen as a decrease in open probability within each sweep. The continued observation of individual openings during the depolarization demonstrates the allosteric nature of CDI. Bottom row: An average of many sweeps demonstrating CDI kinetics. Data reproduced from Yue et al. (1990) with permission from Science.
Independent form of Ca 2+ regulation driven by each lobe of CaM. (A) Whole-cell recordings of CaV1.3 (α1D), using Ca²⁺ and Ba²⁺ as charge carriers, enable visualization of CDI— seen as the increased decay of the Ca²⁺ current (gray) as compared with Ba²⁺ (black). Each lobe of CaM drives distinct forms of CDI. CaM34 isolates Ca²⁺ binding to only the N-lobe of CaM, resulting in CDI with slower kinetics (left) as compared with CaM12 (right), where only the C-lobe remains capable of binding Ca²⁺. Data reproduced from Yang et al. (2006); Copyright 2006 Society for Neuroscience. (B) Cartoon demonstrating the spatial distribution of Ca²⁺-triggering CDI. Under physiological conditions, buffering is low, permitting both a local signal corresponding to Ca²⁺ entry during each channel opening and characterized by large, brief spikes of Ca²⁺ within the nanodomain of the channels (left). High intracellular buffering (e.g., 10 mM BAPTA) restricts Ca²⁺ signals to the large local spikes (middle). The smaller but persistent global Ca²⁺ signal results from the cumulative effect of Ca²⁺ entry through multiple channels (right). Adapted from Tadross et al. (2008) with permission from Cell. (C) Each lobe of CaM responds to a distinct Ca²⁺ signal, with the C-lobe invariably responsive to local Ca²⁺ spikes, while the N-lobe acts as either a global or local Ca²⁺ sensor dependent on the channel subtype and presence of the NSCaTE motif in select channels. NSCaTE, N-terminal spatial Ca²⁺-transforming element.
Physiological impact of inactivation deficits. (A) Current clamp recordings of adult guinea pig ventricular myocytes display significant AP prolongation in response to the elimination of the Ca²⁺-binding sites on CaM N-lobe (CaM34), or both N- and C-lobe (CaM1234, red), but minimal effect when only C-lobe CaM is disrupted (CaM12). Figure reproduced from Alseikhan et al. (2002); Copyright (2002) National Academy of Sciences, USA. (B) Top: Exemplar whole-cell current traces in Ba²⁺ utilizing β1b demonstrate loss of VDI in CaV1.2 channels harboring the G406R TS mutation. Bottom: Exemplar whole-cell current traces in Ca²⁺ (red) and Ba²⁺ (black) utilizing β2a (to reduce VDI) demonstrate a deficit in CDI (seen as the faster decay of the Ca²⁺ current as compared with Ba²⁺) due to G406R. Figure adapted from Dick et al. (2016). (C) Current clamp recordings of induced pluripotent stem cell–derived cardiomyocytes derived from control (black) and TS patients harboring G406R (red). The mutation significantly increases the AP duration (APD) at baseline and reduces the ability of verapamil (v) to shorten the AP. Figure reproduced from Bamgboye et al. (2022b) with permission from Journal of Molecular and Cellular Cardiology.
Inactivation of CaV1 and CaV2 channels

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.


Modeling the effects of thin filament near-neighbor cooperative interactions in mammalian myocardium

January 2025

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29 Reads

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1 Citation

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.



Nociceptor sodium channels shape subthreshold phase, upstroke, and shoulder of action potentials

January 2025

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60 Reads

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.


The differential impacts of equivalent gating-charge mutations in voltage-gated sodium channels

January 2025

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34 Reads

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2 Citations

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.




Drugs exhibit diverse binding modes and access routes in the Nav1.5 cardiac sodium channel pore

January 2025

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78 Reads

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2 Citations

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.


Static HTML page with annotated sequence multi-alignment and protein structure visualization. A screenshot of the browser windows generated for the CLC chloride-transporting protein family is shown. On the left, a section of the multiple protein alignment is captured with the insertion of graphical annotations better explained in Fig. 2. On the right, the legend relative to the color and font style used in the sequence alignment is shown. At the top of the page, it is indicated which pdb file is currently loaded in the molecular visualization box, with the option to show or not Hetatoms.
Details from the CLC HTML output page. (A) Portion of the CLC multi-alignment showing a large number of pathogenic variants in the alpha-helices G and H (indicated as red bars below the sequence alignment) for all CLCs. The color of the residues depends on the electrophysiological transport characteristics of pathogenic mutations. The color code used in this example is as follows: red, loss of function; green, WT-like; orange, reduction of current; yellow, voltage-shifted; blue, gain of function; and pink, not investigated yet. (B) Zoom of the sequence alignment in the region of the hCLC-1 residue A298. Hovering the mouse over A298 invokes appearance of the box containing annotated comments. (C) 3-D structure of hCLC-1 zoomed around the residue A298, shown in stick and colored in pink.
Overview of the ALLIN web interface. View of the three sections of the web interface. (A) Section 1 contains the info field and the two inputs that define the font size and the number of residues per line that will be shown in the multi-sequence alignment. At the top, five buttons are present: to start the generation of the HTML output (“work”), to clean all fields (“Reset all”), and three instruction buttons. (B) The three input fields with examples of a fasta alignment, a set of rules, and protein atomic coordinates. (C) Example of an HTML output script.
ALLIN: A tool for annotation of a protein alignment combined with structural visualization

December 2024

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41 Reads

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.


The calcium-binding protein S100A1 binds to titin’s N2A insertion sequence in a pH-dependent manner

December 2024

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10 Reads

Titin is the third contractile filament in the sarcomere, and it plays a critical role in sarcomere integrity and both passive and active tension. Unlike the thick and thin filaments, which are polymers of myosin and actin, respectively, titin is a single protein that spans from Z-disk to M-line. The N2A region within titin has been identified as a signaling hub for the muscle and is shown to be involved in multiple interactions. The insertion sequence (UN2A) within the N2A region was predicted as a potential binding site for the Ca²⁺-binding protein, S100A1. We demonstrate using a combination of size exclusion chromatography, surface plasmon resonance, and fluorescence resonance energy transfer that S100A1 can bind to the UN2A region. We further demonstrate that this interaction occurs under conditions where calcium is bound to S100A1, suggesting that the conformational shift in S100A1 when calcium binds is important. We also observed a conformational change in UN2A induced by shifts in pH, suggesting that conformational flexibility in UN2A plays a critical role in the interaction with S100A1. These results lead us to propose that the interaction of S100A1 and UN2A might act as a sensor to regulate titin’s function in response to physiological changes in the muscle.


Reduced voltage-activated Ca release flux in muscle fibers from a rat model of Duchenne dystrophy

December 2024

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21 Reads

The potential pathogenic role of disturbed Ca²⁺ homeostasis in Duchenne muscular dystrophy (DMD) remains a complex, unsettled issue. We used muscle fibers isolated from 3-mo-old DMDmdx rats to further investigate the case. Most DMDmdx fibers exhibited no sign of trophic or morphology distinction as compared with WT fibers and mitochondria and t-tubule membrane networks also showed no stringent discrepancy. Under voltage clamp, values for holding current were similar in the two groups, whereas values for capacitance were larger in DMDmdx fibers, suggestive of enhanced amount of t-tubule membrane. The Ca²⁺ current density across the channel carried by the EC coupling voltage sensor (CaV1.1) was unchanged. The maximum rate of voltage-activated sarcoplasmic reticulum (SR) Ca²⁺ release was reduced by 25% in the DMDmdx fibers, with no change in voltage dependency. Imaging resting Ca²⁺ revealed rare spontaneous local SR Ca²⁺ release events with no sign of elevated activity in DMDmdx fibers. Under current clamp, DMDmdx fibers generated similar trains of action potentials as WT fibers. Results suggest that reduced peak amplitude of SR Ca²⁺ release is an inherent feature of this DMD model, likely contributing to muscle weakness. This occurs despite a preserved amount of releasable Ca²⁺ and with no change in excitability, CaV1.1 channel activity, and SR Ca²⁺ release at rest. Although we cannot exclude that fibers from the 3-mo-old animals do not yet display a fully developed disease phenotype, results provide limited support for pathomechanistic concepts frequently associated with DMD such as membrane fragility, excessive Ca²⁺ entry, or enhanced SR Ca²⁺ leak.


Distinct properties and activation of hexameric and heptameric Pannexin 1 channel concatemers

December 2024

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55 Reads

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1 Citation

Pannexin 1 (PANX1) is a member of a topologically related and stoichiometrically diverse family of large pore membrane ion channels that support the flux of signaling metabolites (e.g., ATP) and fluorescent dyes. High-resolution structural analyses have identified PANX1 as a heptamer despite early evidence suggesting that it might be a hexamer. To determine if PANX1 channel activity is supported in both hexameric and heptameric conformations, we examined properties of concatenated PANX1 constructs comprising either six or seven subunits with intact or truncated C-termini (the latter to mimic caspase-cleavage activation). In whole-cell recordings from PANX1-deleted cells, the C-tail-truncated hexameric and heptameric concatemers generated outwardly rectifying PANX1-like currents only after severing the intersubunit linkers. Surprisingly, α1D adrenoceptor stimulation activated constructs with intact or truncated C-tails, even without linker cleavage. In inside-out patches from PANX1-deleted cells, linker cleavage activated C-tail truncated channels derived from either hexameric or heptameric concatemers. The heptamers presented peak unitary conductance and mean open time that was similar to channels assembled from the expression of unlinked single PANX1 subunits and greater than from the hexamers. In addition, the linker-cleaved heptameric concatemers supported greater PANX1-dependent ATP release and TO-PRO-3 uptake than the corresponding hexamers. These data indicate that functional PANX1 channels can be obtained in either hexameric or heptameric conformations and suggest that the distinct unitary properties of heptameric channels are more conducive to large molecule permeation by PANX1; they also suggest that there are distinct structural requirements for C-tail cleavage and receptor-mediated PANX1 activation mechanisms.


Mechanisms regulating mitochondrial Ca 2+ . The MCU is the primary protein responsible for Ca²⁺ uptake into the mitochondria. The energy for Ca²⁺ uptake is provided by the large negative membrane potential that is generated by the electron transport chain (ETC). The ETC is also responsible for generating an alkaline matrix pH. The inwardly directed proton gradient drives the efflux of a number of ions including Na⁺, K⁺, and Ca²⁺. In addition to CHE, Ca²⁺ efflux from the matrix occurs via a CHE. There are also data suggesting a role for Ca²⁺ entry via an electrogenic Ca²⁺/H⁺ exchanger, although this needs confirmation. Much of the mitochondrial Ca²⁺ is buffered within Ca-P granules. The phosphate enters the mitochondria on the phosphate carrier (PiC) in exchange for proton.
How does mitochondrial Ca change during ischemia and reperfusion? Implications for activation of the permeability transition pore

Cardiac ischemia followed by reperfusion results in cardiac cell death, which has been attributed to an increase of mitochondrial Ca²⁺ concentration, resulting in activation of the mitochondrial permeability transition pore (PTP). Evaluating this hypothesis requires understanding of the mechanisms responsible for control of mitochondrial Ca²⁺ in physiological conditions and how they are altered during both ischemia and reperfusion. Ca²⁺ influx is thought to occur through the mitochondrial Ca²⁺ uniporter (MCU). However, with deletion of the MCU, an increase in mitochondrial Ca²⁺ still occurs, suggesting an alternative Ca²⁺ influx mechanism during ischemia. There is less certainty about the mechanisms responsible for Ca²⁺ efflux, with contributions from both Ca²⁺/H⁺ exchange and a Na⁺-dependent Ca²⁺ efflux pathway. The molecular details of both mechanisms are not fully resolved. We discuss this and the contributions of both pathways to the accumulation of mitochondrial Ca²⁺ during ischemia and reperfusion. We further discuss the role of mitochondrial Ca²⁺ in activation of the PTP.




Intrinsic adaptive plasticity in mouse and human sensory neurons

December 2024

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35 Reads

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1 Citation

In response to changes in activity induced by environmental cues, neurons in the central nervous system undergo homeostatic plasticity to sustain overall network function during abrupt changes in synaptic strengths. Homeostatic plasticity involves changes in synaptic scaling and regulation of intrinsic excitability. Increases in spontaneous firing and excitability of sensory neurons are evident in some forms of chronic pain in animal models and human patients. However, whether mechanisms of homeostatic plasticity are engaged in sensory neurons of the peripheral nervous system (PNS) is unknown. Here, we show that sustained depolarization (induced by 24-h incubation in 30 mM KCl) induces compensatory changes that decrease the excitability of mouse and human sensory neurons without directly opposing membrane depolarization. Voltage-clamp recordings show that sustained depolarization produces no significant alteration in voltage-gated potassium currents, but a robust reduction in voltage-gated sodium currents, likely contributing to the overall decrease in neuronal excitability. The compensatory decrease in neuronal excitability and reduction in voltage-gated sodium currents reversed completely following a 24-h recovery period in a normal medium. Similar adaptive changes were not observed in response to 24 h of sustained action potential firing induced by optogenetic stimulation at 1 Hz, indicating the need for prolonged depolarization to drive engagement of this adaptive mechanism in sensory neurons. Our findings show that mouse and human sensory neurons are capable of engaging adaptive mechanisms to regulate intrinsic excitability in response to sustained depolarization in a manner similar to that described in neurons in the central nervous system.


Isoform-specific N-linked glycosylation of NaV channel α-subunits alters β-subunit binding sites

December 2024

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13 Reads

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1 Citation

Voltage-gated sodium channel α-subunits (NaV1.1–1.9) initiate and propagate action potentials in neurons and myocytes. The NaV β-subunits (β1–4) have been shown to modulate α-subunit properties. Homo-oligomerization of β-subunits on neighboring or opposing plasma membranes has been suggested to facilitate cis or trans interactions, respectively. The interactions between several NaV channel isoforms and β-subunits have been determined using cryogenic electron microscopy (cryo-EM). Interestingly, the NaV cryo-EM structures reveal the presence of N-linked glycosylation sites. However, only the first glycan moieties are typically resolved at each site due to the flexibility of mature glycan trees. Thus, existing cryo-EM structures may risk de-emphasizing the structural implications of glycans on the NaV channels. Herein, molecular modeling and all-atom molecular dynamics simulations were applied to investigate the conformational landscape of N-linked glycans on NaV channel surfaces. The simulations revealed that negatively charged sialic acid residues of two glycan sites may interact with voltage-sensing domains. Notably, two NaV1.5 isoform-specific glycans extensively cover the α-subunit region that, in other NaV channel α-subunit isoforms, corresponds to the binding site for the β1- (and likely β3-) subunit immunoglobulin (Ig) domain. NaV1.8 contains a unique N-linked glycosylation site that likely prevents its interaction with the β2 and β4-subunit Ig-domain. These isoform-specific glycans may have evolved to facilitate specific functional interactions, for example, by redirecting β-subunit Ig-domains outward to permit cis or trans supraclustering within specialized cellular compartments such as the cardiomyocyte perinexal space. Further experimental work is necessary to validate these predictions.



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