Journal of General Physiology最新文献

Thermosensation requires the activation of a unique collection of ion channels and receptors that work in concert to transmit thermal information. It is widely accepted that transient receptor potential melastatin 8 (TRPM8) activation is required for normal cold sensing; however, recent studies have illuminated major roles for other ion channels in this important somatic sensation. In addition to TRPM8, other TRP channels have been reported to contribute to cold transduction mechanisms in diverse sensory neuron populations, with both leak- and voltage-gated channels being identified for their role in the transmission of cold signals. Whether the same channels that contribute to physiological cold sensing also mediate noxious cold signaling remains unclear; however, recent work has found a conserved role for the kainite receptor, GluK2, in noxious cold sensing across species. Additionally, cold-sensing neurons likely engage in functional crosstalk with nociceptors to give rise to cold pain. This Review will provide an update on our understanding of the relationship between various ion channels in the transduction and transmission of cold and highlight areas where further investigation is required.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.

ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and demonstrated to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. Later experiments revealed that GrTX-SIA could also inhibit voltage-gated K+ (KV) channel currents via a similar mechanism of action that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na+ (NaV) channels contain homologous structural motifs, we hypothesized that GrTX-SIA could inhibit members of this ion channel family as well. Here, we show that GrTX-SIA can indeed impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Moreover, molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as being a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, our data suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide.

Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle. Two UNet-type semantic segmentation models were built: "Locations" classified pixels as belonging to different regions in the cell; "Granules" identified pixels within granules. From their joint output, a pixel fraction pf was calculated for images from patients positive (MHS) or negative (MHN) to a test for malignant hyperthermia susceptibility. pf was used to derive vf, the volume fraction occupied by granules. The relationship vf (pf) was derived from a simulation of volumes ("baskets") containing virtual granules at realistic concentrations. The simulated granules had diameters matching the real ones, which were measured by adapting a utility devised for calcium sparks. Applying this relationship to the pf measured in images, vf was calculated for every region and patient, and from them a glycogen concentration. The intermyofibrillar spaces and the sarcomeric I band had the highest granular content. The measured glycogen concentration was low enough to allow for a substantial presence of non-granular glycogen. The MHS samples had an approximately threefold lower concentration (significant in a hierarchical test), consistent with earlier evidence of diminished glucose processing in MHS. The AI models and the approach to infer three-dimensional magnitudes from two-dimensional images should be adaptable to other tasks on a variety of images from patients and animal models and different disease conditions.

This Commentary discusses the implications of a recent JGP study (Ríos et al. https://www.doi.org/10.1085/jgp.202413595) demonstrating an AI model to quantify glycogen granules.

L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel β subunits (CaVβ) to promote inhibition of ICa,L. In addition to CaVβ interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to β-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.

The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking.