Journal of General Physiology最新文献

Precise control of Kir2.1 channel gating is essential for maintaining membrane potential and enabling repolarization in excitable cells. Disruption of Kir2.1 function can cause Andersen-Tawil syndrome type 1 (ATS1), a multisystem channelopathy that predisposes patients to ventricular dysrhythmias and increases the risk of sudden cardiac death. Kir2.1 activity depends on interactions with the membrane phospholipid PIP2, and these interactions can be weakened by genetic mutations or posttranslational modifications. Here, we identify a shared mechanism by which hypoxia-induced SUMOylation and a heterozygous ATS1-linked variant, R67Q, independently and cooperatively suppress Kir2.1 function. We found that SUMOylation reduces Kir2.1 current in a stoichiometric manner, with up to two SUMO proteins per channel tetramer diminishing current by ∼24% each. Channels containing heterozygous R67Q subunits are disproportionately sensitive to hypoxic suppression. Inhibiting the SUMO pathway with TAK-981 prevents this suppression and enhances current in both WT and R67Q-containing channels. Further analysis revealed that both SUMOylation and the R67Q mutation reduce the stability of Kir2.1-PIP2 interactions, indicating a convergent gating defect. These findings support a two-hit model of channel dysfunction, in which a genetic variant and an environmental stressor act through a common structural mechanism to impair Kir2.1 gating. By identifying PIP2 destabilization as the point of convergence, this work provides new insight into how stress-sensitive channelopathies arise and suggests that SUMO pathway inhibition may offer a strategy to restore function under adverse physiological conditions.

TRPM1 channels, regulated by mGluR6 at the dendrites of retinal ON bipolar cells (BCs), play a crucial role in visual signal transduction. Both Trpm1 knockout (KO) and mGluR6 KO mice are models of congenital stationary night blindness with grossly normal morphology. However, robust pathological spontaneous oscillations in retinal ganglion cells (RGCs) have been observed in Trpm1 KO retinas but not in mGluR6 KO retinas. We investigated the mechanism underlying these oscillations in the Trpm1 KO retina using whole-cell clamp techniques. We found that inhibitory and excitatory synaptic inputs produced anti-phase oscillations in OFF and ON RGCs, respectively, and that oscillations could be suppressed by blockers targeting the AII amacrine cell (AC) pathway. The rd1 retina, a model for retinitis pigmentosa with severe photoreceptor degeneration, displays similar oscillations to the Trpm1 KO retina. Morphological and immunohistochemical analyses revealed similar alterations in the Trpm1 KO and rd1 retinas when compared to the mGluR6 KO and wild-type retinas: namely, rod BCs (RBCs) in both Trpm1 KO and rd1 retinas showed reduced dendritic TRPM1 labeling and smaller axon terminals. Furthermore, RBCs in the Trpm1 KO retina were significantly hyperpolarized. In silico simulation of the BC-AII AC-RGC network suggests that the reduction of RBC and ON cone BC outputs to AII ACs contributes to RGC oscillations. Our findings suggest that TRPM1 deficiency in ON BCs produces RGC oscillations in association with RBC axon remodeling and reduced ON BC outputs, and may represent a shared circuit mechanism underlying pathological oscillations across different causes of outer retinal diseases.

In this special issue of the Journal of General Physiology (JGP), we bring together a collection of studies that exemplify the multidimensional progress in physiology, pharmacology, and structure-function analysis of voltage-gated sodium (NaV) channels. From computational studies and single-residue mutagenesis to insights into drug interactions and electrophysiological variability, the assembled papers illustrate the richness and continuing momentum of this field.

Cortical spreading depression (CSD) is a transient wave of neuronal and glial depolarization that propagates slowly through the cerebral cortex and is implicated in neurological events such as migraine aura. While glutamate, GABA, and serotonin have established roles in CSD modulation, the contribution of dopaminergic signaling, particularly via D2 receptors (D2Rs), remains unclear. In this study, we examined whether topical cortical application of D2R-targeting agents alters CSD propagation and neuronal activation in vivo. Using a KCl-induced CSD model in anesthetized male Wistar rats, we applied metoclopramide (MCP), raclopride (RCP), and quinpirole (QNP) directly onto the cortex. MCP completely blocked CSD propagation at all time points. RCP and QNP produced opposing, time-dependent effects: RCP initially reduced CSD speed, followed by an increase after prolonged exposure, whereas QNP transiently accelerated propagation at 5 min but suppressed it with longer exposure. These changes were accompanied by alterations in waveform morphology, particularly in the secondary negative deflection. c-Fos immunoreactivity revealed reduced neuronal activation in MCP- and QNP-treated animals, mainly in superficial cortical layers, while RCP showed no significant effect. To support these findings, a reaction-diffusion computational model incorporating drug diffusion, receptor binding kinetics, and excitability parameters successfully reproduced the experimental CSD propagation profiles. Together, these results demonstrate that cortical D2R ligands modulate CSD dynamics and neuronal activation in a ligand-specific and time-dependent manner. This study provides mechanistic insight into how dopaminergic signaling influences cortical excitability and CSD propagation, advancing our understanding of dopamine's role in fundamental neurophysiological processes.

Cantu syndrome (CS) is a rare disease caused by gain-of-function (GOF) mutations of Kir6.1 or SUR2 subunits of ATP-sensitive potassium (KATP) channels. CS patients with SUR2 and Kir6.1 variants display a similar constellation of symptoms, including muscle weakness and fatigue. The effects of CS mutations on skeletal muscle KATP channels, and any consequent direct effects on contractility, are currently unclear. Here, we used two knock-in mouse models of CS, respectively, carrying GOF mutations Kir6.1[V65M] or SUR2[A478V], to assess KATP channel properties and contractility in isolated fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles. Electrophysiological recordings in isolated myofibers showed normal resting potentials, and excised patch-clamp recordings showed normal KATP channel density in both genotypes, but enhanced Mg-nucleotide activation only in SUR2[A478V] fibers, consistent with muscle KATP channels being formed predominantly as complexes of SUR2A and Kir6.2 subunits. Ex vivo testing of isolated SUR2[A478V], but not Kir6.1[V65M], muscles showed an earlier onset of fatigue and a marked intra-tetanic decline of force compared with littermate controls. Importantly, normal contractile behavior was restored ex vivo and in vivo in SUR2[A478V] muscles in the presence of the FDA-approved KATP channel inhibitor glibenclamide, indicating that the increased fatigue of isolated muscles is a direct consequence of overactive sarcolemmal KATP channels. These results shed light on the pathophysiologic relevance of SUR2-dependent KATP channel subunits in skeletal muscle and highlight their role in fatiguing conditions, as well as identifying potential therapeutic benefit of skeletal muscle KATP inhibition in CS.

Cardiac contractility is driven by shortening of ∼2-μm-long, macromolecular assemblies known as sarcomeres. During contraction, the motor protein myosin binds to, and exerts force upon actin filaments, utilizing energy from the hydrolysis of ATP. When not actively contracting, myosin partition into two subpopulations, distinguished by their basal rates of ATP hydrolysis, known as the "Disordered Relaxed" (DRX) and "Super Relaxed" (SRX) states. Additionally, the slower hydrolyzing SRX state has been proposed as a sequestered or "reserve pool" of myosin that do not contribute to contraction but can be recruited for enhanced contractility in response to external stimuli. Thus, the fraction of myosin in the SRX state is thought to reflect the overall regulatory state of the myosin population. In this volume of the Journal of General Physiology, a study by Pilagov et al. explores how the SRX state is regulated by phosphorylation or haploinsufficiency of a key regulatory protein, Myosin Binding Protein-C (MyBP-C). Surprisingly, they found that perturbations of MyBP-C led to a negligible change in the overall abundance of SRX. Instead, they found a rearrangement of SRX myosin throughout the sarcomere - specifically a decrease in SRX in regions of the sarcomere that contain MyBP-C and a compensatory increase in SRX in regions lacking MyBP-C. Their findings suggest that the influence of MyBP-C extends beyond its immediate vicinity and can simultaneously exert both positive and negative effects in a location-specific manner.

The sodium leak channel NALCN, a key regulator of neuronal excitability, associates with three ancillary subunits that are critical for its function: a subunit called FAM155, which interacts with the extracellular regions of the channel, and two cytoplasmic subunits called UNC79 and UNC80. Interestingly, NALCN and FAM155 have orthologous phylogenetic relationships with the fungal calcium channel Cch1 and its subunit Mid1; however, UNC79 and UNC80 have not been reported outside of animals. In this study, we leveraged expanded gene sequence data available for eukaryotes to reexamine the evolutionary origins of NALCN and Cch1 channel subunits. Our analysis corroborates the direct phylogenetic relationship between NALCN and Cch1 and identifies a larger clade of related channels in additional eukaryotic taxa. We also identify homologues of FAM155/Mid1 in Cryptista algae and UNC79 and UNC80 homologues in numerous non-metazoan eukaryotes, including basidiomycete and mucoromycete fungi and the microbial eukaryotic taxa Apusomonadida, Malawimonadida, and Discoba. Furthermore, we find that most major animal lineages, except ctenophores, possess a full complement of NALCN subunits. Comparing structural predictions with the solved structure of the human NALCN complex supports orthologous relationships between metazoan and non-metazoan FAM155/Mid1, UNC79, and UNC80 homologues. Together, our analyses reveal unexpected diversity and ancient eukaryotic origins of NALCN/Cch1 channelosome subunits and raise interesting questions about the functional nature of this channel complex within a broad, eukaryotic context.

Stretch activation (SA) is the delayed increase in force following a rapid stretch and improves muscle performance during repetitive cyclical contractions in insect flight and cardiac muscles. Although historically considered too low to be physiologically relevant in skeletal muscle, our recent work showed that higher phosphate concentrations ([Pi]) increased SA in mouse soleus fibers. These results suggest SA has a role combating fatigue, which increases [Pi], lowers pH, and reduces active calcium concentration ([Ca2+]). To test this, we measured SA during Active, High [Ca2+] Fatigue and Low [Ca2+] Fatigue conditions in myosin heavy chain (MHC) I, IIA, IIX, and IIB fibers from mouse soleus and extensor digitorum longus muscles. In the fast-contracting MHC II fibers, calcium-activated isometric tension (F0) decreased from Active to High [Ca2+] Fatigue to Low [Ca2+] Fatigue, as expected. Remarkably, SA tension (FSA) was not decreased but remained unchanged or increased under High and Low [Ca2+] Fatigue, except for a small decrease in MHC IIB fibers in Low [Ca2+] Fatigue compared with Active. This results in SA's percent contribution to total tension production (FSA/[F0 + FSA]) being much greater (58-114%) under fatiguing conditions in fast-contracting MHC II fibers. The SA tension peak for MHC I fibers was not visibly apparent under either fatigue condition, and the peak was about 20% of MHC II fibers' peaks under active conditions. Our results show SA improves force production under fatiguing conditions in MHC II fibers, which could play an important role in increasing endurance for muscles that are lengthened prior to shortening.

β-cardiac myosin mediates cardiac muscle contraction within the sarcomere by binding to the thin filament in an ATP-powered reaction. This process is highly regulated on a beat-to-beat basis by calcium interactions with the thin filament, but also contractile force is highly regulated by controlling the number of myosins available, resulting in a dynamic reserve. Our goal was to examine the size of this reserve and how it is modulated by cardiac myosin binding protein-C (cMyBP-C). We used single-molecule imaging to determine myosin activity with high spatial resolution by measuring fluorescently tagged ATP molecules binding to and releasing from myosins within the cardiac sarcomere. Three myosin ATPase states were detected: the fastest species was consistent with nonspecific ATP binding to myosin's surface, and the slower two species were consistent with the previously identified DRX and SRX states. The former represents myosins in a state ready to interact with the thin filament, and the latter in a cardiac reserve state with slowed ATPase. We found the cardiac reserve was 46% across the whole sarcomere in porcine myofibrils. Subdividing into the P-, C-, and D-zones revealed the D-zone has the smallest population of reserve heads (44%). Treatment with PKA that phosphorylates cMyBP-C led to a 16% reduction of reserve in the C-zone (where cMyBP-C is found) and a 10% reduction in the P-zone, with an unexpected 15% increase in the D-zone. Interestingly, the changes in SRX myosin head distribution by PKA phosphorylation of cMyBP-C across each subsarcomeric zone mirror the changes we identified in human cardiac myofibrils isolated from a hypertrophic cardiomyopathy patient mutation (MYBPC3-c.772G>A) that exhibits cMyBP-C haploinsufficiency. These results provide novel insights into how the C-zone functions in both porcine and human β-cardiac myosin-containing thick filaments, revealing a possible compensatory change in the D-zone upon altered cMyBP-C phosphorylation and/or haploinsufficiency.