Journal of General Physiology最新文献

Voltage-gated sodium channels underpin electrical signaling in sensory neurons. Their activity is an essential element in the vast majority of pain conditions, making them significant drug targets. Sensory neuron sodium channels play roles not only in afferent signaling but also in a range of efferent regulatory mechanisms. Side effects through actions on other cell types and efferent signaling are thus important issues to address during analgesic drug development. As an example, the human genetic evidence for NaV1.7 as an ideal pain target contrasts with the side effects of NaV1.7 antagonists. In this review, we describe the history and progress toward the development of useful analgesic drugs and the renewed focus on NaV1.8 as a key target in pain treatment. NaV1.8 antagonists alone or in combination with other analgesics are likely to provide new opportunities for pain relief for the vast number of people (about 33% of the population) impacted by chronic pain, particularly present in aging populations.

To study the physiological and pathological mechanisms of muscle, it is crucial to store muscle samples in ways that preserve their properties. Glycerol is commonly used for storage, as it stabilizes muscle proteins, slows enzymatic activity, and minimizes degradation. However, previous studies validating glycerol storage have not examined its effects on passive properties. In this study, mouse extensor digitorum longus (EDL) muscles were stored in 50% glycerol in relaxing solution with protease inhibitors for various durations, then rehydrated in physiological solutions to assess mechanical properties. Active properties remained unchanged, but passive stress was sensitive to glycerol storage, showing a 56.5 ± 13.6% increase after 4 days, and this effect was permanent. The increase was most pronounced at sarcomere lengths, where titin's PEVK segment extension dominates. Using gelsolin, we determined whether the passive stress increase requires the thin filament, which is known to interact with titin's PEVK region. Both glycerol-stored fibers with and without thin filament extraction exhibited increased passive stress, suggesting that the underlying mechanism is intrinsic to titin. Finally, fibers treated with methylglyoxal, a reactive carbonyl and glycating agent that forms cross-links on lysine residues, showed a significant increase in passive stress in fibers stored in relaxing solution but not in glycerol. Thus, glycerol storage elevates passive stress in a titin-specific manner, likely involving lysine residues in the PEVK. Therefore, glycerol storage should be avoided when assessing passive stiffness. We further showed that, for long-term preservation, storage of rapidly frozen muscle at -80°C is a viable option.

Nav1.8 sodium channels are expressed in pain-sensing neurons, and some Nav1.8 inhibitors significantly reduce pain in clinical trials. Several Nav1.8 inhibitors have an unusual state dependence whereby inhibition is relieved by depolarization. We compared the state-dependent action of several Nav1.8 channel inhibitors to test whether inhibition is relieved during action potential (AP) firing under physiological conditions to produce "reverse use dependence." A-887826 inhibition was substantially relieved by AP waveforms applied at 20 Hz at 37°C. In contrast, there was no relief during AP trains with suzetrigine (VX-548) or LTGO-33, even though inhibition could be effectively removed by long, strong depolarizations. These differences were explained by differences in the voltage dependence and kinetics with which the compounds dissociate from depolarized channels and rebind to resting state channels. Suzetrigine required the strongest depolarizations for relief (midpoint +33 mV) and relief was slow (tau >300 ms at +20 mV), so almost no relief occurred during an AP waveform. Relief from A-887826 required weaker depolarizations (midpoint +13 mV) and was much faster, so some relief occurred during each AP waveform and accumulated during 20-Hz trains. LTGO-33 required the weakest depolarizations for relief (midpoint -11 mV) and relief was even faster than for A-887826, but reinhibition between AP waveforms was far faster than for A-887826, so that relief did not accumulate during AP trains at 20 Hz. The results show that, unlike A-887826, there is no use-dependent relief of inhibition by suzetrigine or LTGO-33 with physiological voltage waveforms at physiological temperatures, but each for different reasons.

In cardiac muscle, regulation of actin polymerization at the thin filament pointed end is controlled by two structurally similar but functionally antagonistic proteins, leiomodin-2 and tropomodulin-1. Both proteins contain tropomyosin-binding site 1, which is essential for their recruitment to the pointed end. Using circular dichroism, we determined changes in melting temperatures (ΔTm) for complexes of tropomyosin and leiomodin-2 fragments containing several hypomorphic mutations, which moderately affect binding to tropomyosin. We ran molecular dynamics simulations for the complexes and calculated standard Gibbs free energies of binding, which we found to strongly correlate with the ΔTm. We found that the E34Q mutation in leiomodin-2 resulted in a decrease in the melting temperature of the complex of tropomyosin and leiomodin-2 fragments, indicating a decrease in the affinity of leiomodin-2 for tropomyosin. Although modest, this change in in vitro affinity made leiomodin-2 a weaker competitor for tropomyosin than tropomodulin-1 in cardiomyocytes. This mutation significantly reduced the ability of leiomodin-2 to displace tropomodulin-1 at thin filament pointed ends and affected the ability of leiomodin-2 to elongate thin filaments. Our results highlight the essential role of the tropomyosin-binding site in the dynamic equilibrium between tropomodulin-1 and leiomodin-2 at the pointed end of thin filaments. Our data also suggest the potential use of the correlation between ΔTm and the modeled standard Gibbs free energies of binding to predict changes in the stability of complexes between tropomyosin and leiomodin or tropomodulin isoforms.

JGP study (Han et al. https://doi.org/10.1085/jgp.202413729) reveals that glycerol storage increases the titin-based stiffness of muscle fibers, suggesting that this commonly used method should be avoided by researchers interested in the passive properties of muscle.

Cytosolic N termini of several BK channel β regulatory subunits mediate rapid inactivation. However, in contrast to Kv channels, inactivation does not occur via a simple, open-channel block mechanism, but involves two steps, an association step in which ion permeation is maintained (O*), then followed by inactivation (I). To produce inactivation, BK β subunit N termini enter the central cavity through a lateral entry pathway ("side portal") separating the transmembrane pore-gate domain and cytosolic gating ring. Comparison of BK conformations reveals an aqueous pathway into the central cavity in the open structure, while in the closed structure, three sequential basic residues (R329K330K331) in the C-linker just following S6 occlude central cavity access. We probed the impact of mutations of the RKK motif (RKK3Q, RKK3E, and RKK3V) on inactivation mediated by the β3a N terminus. All three RKK-mutated constructs differentially reduce depolarization-activated outward current, prolong β3a-mediated tail current upon repolarization, and produce a persistent inward current at potentials down to -240 mV. With depolarization, channels are driven into O*-I inactivated states, and upon repolarization, slow tails and persistent inward currents reflect slow changes in O*-I occupancy. However, evaluation of closed-state occupancy prior to depolarization and at the end of slow tails reveals that some fraction of closed states at negative potentials correspond to resting closed states in voltage-independent equilibrium with N-terminal-occluded closed states. Thus, disruption of the RKK triplet both stabilizes the β3a N terminus in its position of inactivation and permits access of that N terminus to its blocking position in closed states.

GJC2 encodes connexin 47 (Cx47), a gap junction protein expressed by oligodendrocytes that forms gap junction channels (GJCs) between adjacent oligodendrocytes (or astrocytes, via heterotypic Cx47-Cx43 GJCs). Autosomal recessive mutations of GJC2 lead to at least three central nervous system phenotypes: Pelizaeus-Merzbacher-like disease 1 (PMLD1), spastic paraparesis 44 (SPG44), and a minimal leukodystrophy. Here, we describe the clinical, functional, and molecular effects of two mutations in GJC2, p.G40S, and p.R244P, identified in two different families with GJC2-related disorders. Expressed exogenously, p.G40S forms GJC plaques like WT but does not functionally couple with WT nor with Cx43. p.R244P also fails to demonstrate functional coupling. Moreover, plaque formation is absent, concomitant with intracellular connexin accumulation. When the two mutants are co-expressed in a compound heterozygous state, plaques form, but no GJC coupling is detected in any configuration. MD simulations demonstrate that p.G40S modifies secondary structure of the pore-lining α-helix, disrupting supersecondary interactions with the N-terminal helix and predicting channel closure. p.R244P simulations are characterized by partial loss of the extracellular β-sheet domains and a marked reduction of electrostatic interactions between the connexin and lipid headgroups of the plasma membrane, suggesting pathways by which p.R244P mutation impairs GJC formation. This combination of in vitro assays and molecular simulations provides mechanistic insight into the pathogenesis of GJC2-related disease.

Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with deficits in motor coordination and cognitive function. Mice lacking iFGF14 (Fgf14-/-) display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to a hyperpolarizing shift in the voltage-dependence of voltage-gated Na+ (Nav) current steady-state inactivation. Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal pyramidal neurons. Current-clamp recordings from CA1 pyramidal neurons in acute in vitro slices, however, revealed that evoked repetitive firing rates were higher in Fgf14-/- than in wild type (WT) cells. Also, in contrast with Purkinje neurons, voltage-clamp recordings demonstrated that the loss of iFGF14 did not affect the voltage dependence of steady-state inactivation of the Nav currents in CA1 pyramidal neurons. In addition, in contrast with results reported for neonatal (rat) hippocampal pyramidal neurons in dissociated cell culture, immunohistochemical experiments revealed that loss of iFGF14 does not disrupt the localization or alter the normalized distribution of α-Nav1.6 or α-ankyrin G labeling along the axon initial segments (AIS) of mature hippocampal CA1 neurons in situ. However, the integrated intensities of α-Nav1.6 labeling were significantly higher along the AIS of Fgf14-/-, compared with WT, adult hippocampal CA1 pyramidal neurons, consistent with the marked increase in the excitability of CA1 neurons with the loss of iFGF14.

Metaphorical language is ubiquitous throughout the life sciences, with, for example, molecules forming chains which define genetic blueprints for the development of cells and ultimately the gates in the channels forming the subject of this special issue. Indeed, metaphor is a fundamental component of scientific discourse and influences how science is both communicated and understood across all levels of expertise. This article, written for readers without a background in linguistics, first provides a brief introduction to the mechanisms of scientific metaphor and then illustrates its productive application to the sodium channel fast inactivation mechanism.

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.