Channels (Austin, Tex.)最新文献

Accumulation of abdominal visceral adipose tissue (VAT) is a major risk factor for cardiovascular disease. Obesity-induced endothelial dysfunction is a precursor to severe disease, and we and others have shown that arteries embedded in VAT, but not subcutaneous adipose tissue, exhibit robust endothelial dysfunction. Using a mouse model of diet-induced obesity, we recently linked VAT from obese mice to the impairment of endothelial Kir2.1, a critical regulator of endothelial function. However, the mechanism by which VAT impairs Kir2.1 is unclear. As Kir2.1 impairment is dependent on endothelial CD36, we hypothesized that lipolytic VAT induces Kir2.1 impairment through fatty acids (FA). To test this, we first treated endothelial cells with palmitic acid (PA) to determine whether the addition of exogenous FAs recapitulated our original finding of Kir2.1 dysfunction when challenged with VAT. PA inhibited Kir2.1 assessed via whole-cell patch-clamp electrophysiology, an effect that was dependent on endothelial CD36. To determine whether inhibiting VAT lipolysis prevents Kir2.1 dysfunction in the presence of VAT in obese mice and humans, VAT was pretreated with small molecule inhibitors of adipose triglyceride lipase prior to incubating endothelial cells with adipose tissue. This approach also prevented VAT-induced impairment of endothelial Kir2.1 suggesting that VAT-derived FAs may play a role. Furthermore, inhibition of lipolysis in the VAT of obese mice and humans significantly reduced endothelial FA uptake, similar to that observed when CD36 was downregulated. These findings advance our understanding of the relationship between VAT and endothelial Kir2.1 impairment and place VAT-derived FAs as potential paracrine mediators.

A growing body of preclinical evidence indicates that the inhibition of voltage-gated Cav1.3 L-type Ca²⁺ channels could be a therapeutic concept for the therapy of treatment-resistant hypertension, spinal injury and for neuroprotection in early Parkinson's disease (PD). However, available Ca²⁺-channel blockers are potent inhibitors of vascular Cav1.2 L-type channels which can cause low blood pressure as an adverse drug reaction. Therefore, Cav1.3-selective inhibitors are needed to further investigate the therapeutic potential of Cav1.3 as drug target in vivo. The bicyclic diterpene alcohol sclareol has recently been reported to exert neuroprotective properties in a mouse PD model by blocking Cav1.3 L-type channels. This study investigates the proposed Cav1.3-selectivity of sclareol compared to Cav1.2 and to other voltage-gated Ca²⁺ channels in whole-cell patch-clamp experiments. Various stimulation protocols, including dopamine neuron-like firing patterns show that sclareol is neither a subtype-selective nor a potent blocker of heterologously expressed Cav1.3 and inhibits also Cav2.3 channels. Therefore, the contribution of Cav1.3 channel inhibition for the previously reported neuroprotective effects of sclareol in a mouse PD model remains unclear. In addition, cinnarizine, a vertigo therapeutic also under investigation for inhibition of Cav1.3-mediated aldosterone-secretion, inhibits Cav1.3 channels in a frequency-dependent manner, but also without relevant selectivity with respect to Cav1.3.

Kir (inwardly rectifying potassium) channels that play key roles in maintaining potassium homeostasis, neuronal excitability, and osmoregulation have been cloned and characterized in a variety of insects. In Drosophila melanogaster, three Kir channels (dKir1 dKir2, and dKir3) have been cloned and characterized, and share significant homology with mammalian Kir channels. The dKir channels are essential for various developmental processes, such as wing patterning, by modulating bone morphogenetic protein signaling pathways. Electrophysiological studies have confirmed that Drosophila Kir channels function in a way analogous to their mammalian counterparts, indicating that their roles in cellular and developmental signaling have been evolutionarily conserved. Several Kir channels have also been identified and characterized in mosquitoes (Aedes aegypti and Anopheles gambiae). Interestingly, insect Kir channel orthologs cluster into three gene "clades" or subfamilies (Kir1, Kir2, Kir3) that are distinct from mammal Kir channels based on sequence comparisons. Insect Kir channel paralogs range from two to eight Kir channel genes per species genome representing separate gene duplication events. These differences may be attributed to distinct physiological adaptations associated with their respective taxonomic groups. The honeybee Apis mellifera genome contains two Kir channel genes, AmKir1 and AmKir2, producing six Kir channel isoforms via alternative splicing, which have been cloned and expressed in heterologous systems to study their electrophysiological properties. This review provides a comprehensive overview of current knowledge about Kir channel structures, activities, and gating as well as of their roles in insects, including evolutionary genomic aspects, molecular biology, physiological roles, and pharmacological targeting.

Potassium ion channel (K⁺ channel) is a crucial transmembrane protein found on cell membranes that plays a pivotal role in regulating various physiological processes such as cell membrane potential, action potential formation, and cellular excitability. Diabetes, a chronic metabolic disorder characterized by elevated blood glucose levels, can cause abnormal changes in the sensitivity and functioning of K⁺ channels over time. This can lead to an increase in intracellular K⁺ and Ca²⁺, disrupting normal cellular function and metabolism and resulting in a range of physiological and metabolic issues. Recent studies have uncovered the collaborative relationship between K⁺ channels auxiliary SUR1 and Kir6.2 gating, as well as the impact of K+ channel mutations such as KCNK11 Leu114Pro, KCNQ1Arg397Trp, KCNJ11Arg136Cys, KCNK16 Leu114Pro, and KCNMA1 Gly356Arg on diabetes mellitus and associated complications. Specifically, issues such as impaired cardiac repolarization, K_ATP, Kir, TALK, and K_V channel remodeling and a higher risk of arrhythmia have been emphasized. Furthermore, structural and dysfunctional K⁺ channels (K_Ca, K_V and Kir) can also affect the function of vascular endothelial and smooth muscle cells, leading to impaired vasomotor function, abnormal cell growth, and increased inflammation. These abnormalities can result in cardiovascular damage and lesions, and increase the risk of cardiovascular disease in diabetic individuals. These findings serve as a crucial foundation for a better understanding and addressing cardiovascular issues in patients with diabetes. Moreover, different drugs and treatments targeting the K⁺ channel may yield varying effects, offering promising prospects for preventing and managing diabetes and its related complications.

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.