Journal of General Physiology最新文献

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.

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.

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.

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.

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 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 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.

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.

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

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.