Channels (Austin, Tex.)最新文献

The hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) gene has been reported to regulate the spontaneous depolarization of sinoatrial node cells. A novel HCN4 mutation (c.2036 G>A) may lead to sick sinus syndrome. The green fluorescent protein (GFP) and either the wild-type (WT) or C679Y mutant (mut) were co-transfected into HEK293 cells to investigate the impact of the mutation on HCN4 channel function. The whole-cell patch-clamp approach was utilized to record HCN4 currents. According to electrophysiological recording, the current amplitude and density generated by mut-C679Y HCN4 channels were much lower than those generated by WT channels. HCN4 channel current activation was not significantly affected by the C679Y mutation. Because of the little current, analyzing the mut channel deactivation kinetic was challenging. Thus, we have identified a novel HCN4 gene mutation that is connected to bradycardia, left ventricular noncompaction, and diverse valve-related heart conditions.

Microglia, the central nervous system (CNS) resident immune cells, are pivotal in regulating neurodevelopment, maintaining neural homeostasis, and mediating neuroinflammatory responses. Recent research has highlighted the importance of mechanotransduction, the process by which cells convert mechanical stimuli into biochemical signals, in regulating microglial activity. Among the various mechanosensitive channels, Piezo1 has emerged as a key player in microglia, influencing their behavior under both physiological and pathological conditions. This review focuses on the expression and role of Piezo1 in microglial cells, particularly in the context of neuroinflammation and tumorigenesis. We explore how Piezo1 mediates microglial responses to mechanical changes within the CNS, such as alterations in tissue stiffness and fluid shear stress, which are common in conditions like multiple sclerosis, Alzheimer's disease, cerebral ischemia, and gliomas. The review also discusses the potential of targeting Piezo1 for therapeutic intervention, given its involvement in the modulation of microglial activity and its impact on disease progression. This review integrates findings from recent studies to provide a comprehensive overview of Piezo1's mechanistic pathways in microglial function. These insights illuminate new possibilities for developing targeted therapies addressing CNS disorders with neuroinflammation and pathological tissue mechanics.

Mutations in KCNQ2 are linked to various neurological disorders, including neonatal-onset epilepsy. The severity of these conditions often correlates with the mutation's location and the biochemical properties of the altered amino acid side chains. Two mutations affecting aspartate at position 212 (D212) in the S4-S5 linker of KCNQ2 have been identified. Interestingly, while the charge-conserved D212E mutation leads to severe neonatal-onset developmental and epileptic encephalopathy (DEE), the more dramatic substitution to glycine (D212G) results in self-limited familial neonatal epilepsy (SLFNE), a much milder pathology. To elucidate the underlying mechanisms, we performed electrophysiological studies and in silico simulations to investigate these mutations' biophysical and structural effects. Our findings reveal that the D212E mutation stabilizes the channel in the voltage sensor down-state and destabilizes the up-state, leading to a rightward shift in the voltage-dependent activation curve, slower activation kinetics, and accelerated deactivation kinetics. This disruption in KCNQ2 voltage sensitivity persists even in the more physiologically relevant KCNQ2/3 heterotetrameric channels. In contrast, the D212G mutation primarily destabilizes the up-state, but its impact on voltage sensitivity is significantly reduced in KCNQ2/3 heterotetrameric channels. These findings provide key insights into the biophysical and structural basis of KCNQ2 D212 mutations and their contribution to epilepsy-related symptoms, offering a clearer understanding of how these mutations drive the varied clinical outcomes observed in patients.

Induced pluripotent stem cell (iPSC)-derived motor neurons provide a powerful platform for studying motor neuron diseases. These cells enable human-specific modeling of disease mechanisms and high-throughput drug screening. While commercially available iPSC-derived motor neurons offer a convenient alternative to time-intensive differentiation protocols, their electrophysiological properties and maturation require comprehensive evaluation to validate their utility for research and therapeutic applications. In this study, we characterized the electrophysiological properties of commercially available iPSC-derived motor neurons. Immunofluorescence confirmed the expression of motor neuron-specific biomarkers, indicating successful differentiation and maturation. Electrophysiological recordings revealed stable passive membrane properties, maturation-dependent improvements in action potential kinetics, and progressive increases in repetitive firing. Voltage-clamp analyses confirmed the functional expression of key ion channels, including high- and low-voltage-activated calcium channels, TTX-sensitive and TTX-insensitive sodium channels, and voltage-gated potassium channels. While the neurons exhibited hallmark features of motor neuron physiology, high input resistance, depolarized resting membrane potentials, and limited firing capacity suggest incomplete electrical maturation. Altogether, these findings underscore the potential of commercially available iPSC-derived motor neurons as a practical resource for MND research, while highlighting the need for optimized protocols to support prolonged culture and full maturation.

Seventy-five unique variants in the KCNMA1 gene have been identified from individuals with neurological disorders. However, variant pathogenicity and evidence for disease causality are lacking in most cases. In this study, the KCNMA1 variants N999S and E656A (rs886039469 and rs149000684, respectively) were investigated from two individuals presenting with neurological disorders. N999S was previously shown to produce strong gain-of-function (GOF) changes in homomeric BK channel properties in vitro and is found as a heterozygous allele associated with epilepsy and paroxysmal dyskinesia in humans. Although its pathogenicity has been demonstrated in heterozygous animal models, the GOF classification for N999S has not been validated in a heterozygous patient-derived tissue. Conversely, the GOF pathogenicity for E656A is based solely on homomeric channels expressed in vitro and is inconclusive. For either variant, the properties of single heterozygous channels and allele expression is unknown. In this study, we profiled the wild-type and mutant KCNMA1 transcripts from primary human skin fibroblasts of heterozygous patients and unaffected controls and performed patch-clamp electrophysiology to characterize endogenous BK channel current properties. GOF gating was observed in single BK channel recordings from both channel types. Fibroblasts from the individual harboring the E656A variant showed decreases in the number of BK channels detected and E656A-containing transcripts compared to controls. These results show that single BK channels can be reliably detected in primary fibroblasts obtained from human skin biopsies, suggesting their utility for establishing variant pathogenicity, and reveal the BK channel expression and functional changes associated with two heterozygous patient genotypes.

K⁺-dependent Na⁺/Ca²⁺ exchanger proteins (NCKX) are members of the CaCA superfamily with critical roles in vision, skin pigmentation, enamel formation, and neuronal functions. Despite their importance, the structural pathways governing cation transport remain unclear. To address this, we conducted a systematic study using cysteine scanning mutagenesis of human NCKX2 combined with the thiol-modifying reagents MTSET and MTSEA to probe the accessibility and functional significance of specific residues. We used homology models of outward-facing and inward-facing NCKX2 states and molecular dynamics (MD) simulations to compare and investigate residue accessibility in human NCKX2 based on the published structures of the archaeal NCK_Mj Na⁺/Ca²⁺ exchanger and the human NCX1 Na⁺/Ca²⁺ exchanger. Mutant NCKX2 proteins expressed in HEK293 cells revealed diverse effects of MTSET and MTSEA on Ca²⁺ transport. Of the 146 cysteine substitutions analyzed, 35 exhibited significant changes in Ca²⁺ transport activity upon treatment with MTSET, with 16 showing near-complete inhibition and six demonstrating increased activity. Residues within the cation binding sites and extracellular access channels were sensitive to modification, consistent with their critical role in ion transport, whereas intracellular residues showed minimal accessibility to MTSET but were inhibited by membrane-permeable MTSEA. Water accessibility maps from MD simulations corroborated these findings, providing a high-resolution view of water-accessible pathways. This study provides a comprehensive structural and functional map of NCKX2 ion access pathways, offering insights into the molecular basis of ion selectivity and transport. These findings highlight the key residues critical for cation binding and transport, advancing our understanding of the structural dynamics of NCKX2.

The visual process begins with photon detection in photoreceptor outer segments within the retina, which processes light signals before transmission to the thalamus and visual cortex. Cav1.4 L-type calcium channels play a crucial role in this process, and dysfunction of these channels due to pathogenic variants in corresponding genes leads to specific manifestations in visual impairments. This review explores the journey from basic research on Cav1.4 L-type calcium channel complexes in retinal physiology and pathophysiology to their potential as gene therapy targets. Moreover, we provide a concise overview of key findings from studies using different animal models to investigate retinal diseases. It will critically examine the constraints these models present when attempting to elucidate retinal channelopathies. Additionally, the paper will explore potential strategies for addressing Cav1.4 channel dysfunction and discuss the current challenges facing gene therapy approaches in this area of research.

In the European Union, urinary incontinence (UI) affects 45% of adults during their lifetime, representing a major clinical and socio-economic burden. Failure of urethral smooth muscle (USM) to contract normally (hypo or hypercontractility) contributes to UI symptoms such as urine leakage during bladder filling or inability to urinate due to obstruction. Adequate UI treatments are lacking, partially due to a lack in understanding of cellular mechanisms underlying USM contraction. USM contractions rely on Ca²⁺ signaling in urethral smooth muscle cells (USMC), resulting from Ca²⁺ release from internal stores and Ca²⁺ influx from extracellular sources, such as voltage-gated L-type Ca²⁺ channels or store-operated Ca²⁺ entry (SOCE) channels. L-type Ca²⁺ channel inhibitors have inconsistent effects on urethral contractions across species, including humans, and thus solely targeting this pathway may be insufficient to modulate USM contractility. Recent animal experiments suggest SOCE mediated by Orai-STIM proteins is a critical determinant of Ca²⁺ signaling in USMC, maintaining regenerative Ca²⁺ release from internal stores, and thus may be a targetable pathway for influencing USM contractility. In this review, we highlight evidence suggesting SOCE as critical for Ca²⁺ signaling in USMC from multiple species and propose possible mechanisms for how this occurs at the cellular level.

Increased expression of K_Ca3.1 has been found in vascular smooth muscle cells (SMC), macrophages, and T cells in atherosclerotic lesions from humans and mice. Pharmacological inhibition of K_Ca3.1 in limiting atherosclerosis has been demonstrated in mice and pigs, however direct, loss-of-function, i.e. gene silencing, studies are absent. Therefore, we generated K_Ca3.1^-/-Apoe^-/- (DKO) mice and assessed lesion development in the brachiocephalic artery (BCA) of DKO versus Apoe^-/- mice on a Western diet for 3 months. In BCAs of DKO mice, lesion size and relative stenosis were reduced by ~70% compared to Apoe^-/- mice, with no effect on medial or lumen area. Additionally, DKO mice exhibited a significant reduction in macrophage content within plaques compared to Apoe^-/- mice, independent of sex. In vitro migration assays showed a significant reduction in migration of bone marrow-derived macrophages (BMDMs) from DKO mice compared to those from Apoe^-/- mice. In vitro experiments using rat aortic smooth muscle cells revealed inhibition of PDGF-BB-induced MCP1/Ccl2 expression upon K_Ca3.1 inhibition, while activation of K_Ca3.1 further enhanced MCP1/Ccl2 expression. Both in vivo and in vitro analyses showed that silencing K_Ca3.1 had no significant effect on the collagen content of plaque. RNAseq analysis of BCA samples from DKO and Apoe^-/- mice revealed PPAR-dependent signaling as a potential key mediator of the reduction in atherosclerosis due to K_Ca3.1 silencing. Overall, this study provides the first genetic evidence that K_Ca3.1 is a critical regulator of atherosclerotic lesion development and composition and provides novel mechanistic insight into the link between K_Ca3.1 and atherosclerosis.

N-methyl-D-aspartate receptors (NMDARs) are heterotetrameric ion channels that play crucial roles in brain function. Among all the NMDAR subtypes, GluN1-N3 receptors exhibit unique agonist binding and gating properties. Unlike "conventional" GluN1-N2 receptors, which require both glycine and glutamate for activation, GluN1-N3 receptors are activated solely by glycine. Furthermore, GluN1-N3 receptors display faster desensitization, reduced Ca²⁺ permeability, and lower sensitivity to Mg²⁺ blockage compared to GluN1-N2 receptors. Due to these characteristics, GluN1-N3 receptors are thought to play critical roles in eliminating redundant synapses and pruning spines in early stages of brain development. Recent studies have advanced pharmacological tools for specifically targeting GluN1-N3 receptors and provided direct evidence of these glycine-activated excitatory receptors in native brain tissue. The structural basis of GluN1-N3 receptors has also been elucidated through cryo-EM and artificial intelligence. These findings highlight that GluN1-N3 receptors are not only involved in essential brain functions but also present potential targets for drug development.