Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine.

Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels.

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

Dissection of the structure-function relationship of Nav channels.

Voltage-gated sodium channels (Nav) undergo conformational shifts in response to membrane potential changes, a mechanism known as the electromechanical coupling. To delineate the structure-function relationship of human Nav channels, we have performed systematic structural analysis using human Nav1.7 as a prototype. Guided by the structural differences between wild-type (WT) Nav1.7 and an eleven mutation-containing variant, designated Nav1.7-M11, we generated three additional intermediate mutants and solved their structures at overall resolutions of 2.9-3.4 Å. The mutant with nine-point mutations in the pore domain (PD), named Nav1.7-M9, has a reduced cavity volume and a sealed gate, with all voltage-sensing domains (VSDs) remaining up. Structural comparison of WT and Nav1.7-M9 pinpoints two residues that may be critical to the tightening of the PD. However, the variant containing these two mutations, Nav1.7-M2, or even in combination with two additional mutations in the VSDs, named Nav1.7-M4, failed to tighten the PD. Our structural analysis reveals a tendency of PD contraction correlated with the right shift of the static inactivation I-V curves. We predict that the channel in the resting state should have a "tight" PD with down VSDs.

Cryo-EM structure of human voltage-gated sodium channel Nav1.6.

Voltage-gated sodium channel Nav1.6 plays a crucial role in neuronal firing in the central nervous system (CNS). Aberrant function of Nav1.6 may lead to epilepsy and other neurological disorders. Specific inhibitors of Nav1.6 thus have therapeutic potentials. Here we present the cryo-EM structure of human Nav1.6 in the presence of auxiliary subunits ß1 and fibroblast growth factor homologous factor 2B (FHF2B) at an overall resolution of 3.1 Å. The overall structure represents an inactivated state with closed pore domain (PD) and all "up" voltage-sensing domains. A conserved carbohydrate-aromatic interaction involving Trp302 and Asn326, together with the ß1 subunit, stabilizes the extracellular loop in repeat I. Apart from regular lipids that are resolved in the EM map, an unprecedented Y-shaped density that belongs to an unidentified molecule binds to the PD, revealing a potential site for developing Nav1.6-specific blockers. Structural mapping of disease-related Nav1.6 mutations provides insights into their pathogenic mechanism.

Dual-pocket inhibition of Nav channels by the antiepileptic drug lamotrigine.

Voltage-gated sodium (Nav) channels govern membrane excitability, thus setting the foundation for various physiological and neuronal processes. Nav channels serve as the primary targets for several classes of widely used and investigational drugs, including local anesthetics, antiepileptic drugs, antiarrhythmics, and analgesics. In this study, we present cryogenic electron microscopy (cryo-EM) structures of human Nav1.7 bound to two clinical drugs, riluzole (RLZ) and lamotrigine (LTG), at resolutions of 2.9 Å and 2.7 Å, respectively. A 3D EM reconstruction of ligand-free Nav1.7 was also obtained at 2.1 Å resolution. RLZ resides in the central cavity of the pore domain and is coordinated by residues from repeats III and IV. Whereas one LTG molecule also binds to the central cavity, the other is found beneath the intracellular gate, known as site BIG. Therefore, LTG, similar to lacosamide and cannabidiol, blocks Nav channels via a dual-pocket mechanism. These structures, complemented with docking and mutational analyses, also explain the structure-activity relationships of the LTG-related linear 6,6 series that have been developed for improved efficacy and subtype specificity on different Nav channels. Our findings reveal the molecular basis for these drugs' mechanism of action and will aid the development of novel antiepileptic and pain-relieving drugs.

Unwinding and spiral sliding of S4 and domain rotation of VSD during the electromechanical coupling in Nav1.7.

Voltage-gated sodium (Nav) channel Nav1.7 has been targeted for the development of nonaddictive pain killers. Structures of Nav1.7 in distinct functional states will offer an advanced mechanistic understanding and aid drug discovery. Here we report the cryoelectron microscopy analysis of a human Nav1.7 variant that, with 11 rationally introduced point mutations, has a markedly right-shifted activation voltage curve with V1/2 reaching 69 mV. The voltage-sensing domain in the first repeat (VSDI) in a 2.7-Å resolution structure displays a completely down (deactivated) conformation. Compared to the structure of WT Nav1.7, three gating charge (GC) residues in VSDI are transferred to the cytosolic side through a combination of helix unwinding and spiral sliding of S4I and ∼20° domain rotation. A conserved WNФФD motif on the cytoplasmic end of S3I stabilizes the down conformation of VSDI. One GC residue is transferred in VSDII mainly through helix sliding. Accompanying GC transfer in VSDI and VSDII, rearrangement and contraction of the intracellular gate is achieved through concerted movements of adjacent segments, including S4-5I, S4-5II, S5II, and all S6 segments. Our studies provide important insight into the electromechanical coupling mechanism of the single-chain voltage-gated ion channels and afford molecular interpretations for a number of pain-associated mutations whose pathogenic mechanism cannot be revealed from previously reported Nav structures.

Structural basis for high-voltage activation and subtype-specific inhibition of human Nav1.8.

The dorsal root ganglia-localized voltage-gated sodium (Nav) channel Nav1.8 represents a promising target for developing next-generation analgesics. A prominent characteristic of Nav1.8 is the requirement of more depolarized membrane potential for activation. Here we present the cryogenic electron microscopy structures of human Nav1.8 alone and bound to a selective pore blocker, A-803467, at overall resolutions of 2.7 to 3.2 Å. The first voltage-sensing domain (VSDI) displays three different conformations. Structure-guided mutagenesis identified the extracellular interface between VSDI and the pore domain (PD) to be a determinant for the high-voltage dependence of activation. A-803467 was clearly resolved in the central cavity of the PD, clenching S6IV. Our structure-guided functional characterizations show that two nonligand binding residues, Thr397 on S6I and Gly1406 on S6III, allosterically modulate the channel's sensitivity to A-803467. Comparison of available structures of human Nav channels suggests the extracellular loop region to be a potential site for developing subtype-specific pore-blocking biologics.

Structure of human Nav1.5 reveals the fast inactivation-related segments as a mutational hotspot for the long QT syndrome.

Nav1.5 is the primary voltage-gated Na+ (Nav) channel in the heart. Mutations of Nav1.5 are associated with various cardiac disorders exemplified by the type 3 long QT syndrome (LQT3) and Brugada syndrome (BrS). E1784K is a common mutation that has been found in both LQT3 and BrS patients. Here we present the cryo-EM structure of the human Nav1.5-E1784K variant at an overall resolution of 3.3 Å. The structure is nearly identical to that of the wild-type human Nav1.5 bound to quinidine. Structural mapping of 91- and 178-point mutations that are respectively associated with LQT3 and BrS reveals a unique distribution pattern for LQT3 mutations. Whereas the BrS mutations spread evenly on the structure, LQT3 mutations are clustered mainly to the segments in repeats III and IV that are involved in gating, voltage-sensing, and particularly inactivation. A mutational hotspot involving the fast inactivation segments is identified and can be mechanistically interpreted by our "door wedge" model for fast inactivation. The structural analysis presented here, with a focus on the impact of mutations on inactivation and late sodium current, establishes a structure-function relationship for the mechanistic understanding of Nav1.5 channelopathies.

Comparative structural analysis of human Nav1.1 and Nav1.5 reveals mutational hotspots for sodium channelopathies.

Among the nine subtypes of human voltage-gated sodium (Nav) channels, the brain and cardiac isoforms, Nav1.1 and Nav1.5, each carry more than 400 missense mutations respectively associated with epilepsy and cardiac disorders. High-resolution structures are required for structure-function relationship dissection of the disease variants. We report the cryo-EM structures of the full-length human Nav1.1-ß4 complex at 3.3 Å resolution here and the Nav1.5-E1784K variant in the accompanying paper. Up to 341 and 261 disease-related missense mutations in Nav1.1 and Nav1.5, respectively, are resolved. Comparative structural analysis reveals several clusters of disease mutations that are common to both Nav1.1 and Nav1.5. Among these, the majority of mutations on the extracellular loops above the pore domain and the supporting segments for the selectivity filter may impair structural integrity, while those on the pore domain and the voltage-sensing domains mostly interfere with electromechanical coupling and fast inactivation. Our systematic structural delineation of these mutations provides important insight into their pathogenic mechanism, which will facilitate the development of precise therapeutic interventions against various sodium channelopathies.

Effect of hospital elder life program on the incidence of delirium: A systematic review and meta-analysis of clinical trials.

OBJECTIVE: This meta-analysis aims to investigate the effect of the Hospital Elder Life Program (HELP) on the incidence of delirium, delirium scores, length of hospital stay, and incidence of falls. METHODS: Four databases (PubMed, Embase, Cochrane Library, and Web of Science) were searched from inception until January 18, 2024. The search specifically targeted randomized controlled trials (RCTs). Two independent researchers conducted literature screening, quality assessment, and data extraction. The meta-analysis was performed using Review Manager 5.4.1 and Stata 15.1 software. RESULTS: The final analysis included a total of 9 RCTs with 2583 patients. The findings from the meta-analysis indicated that HELP was found to considerably reduce the incidence of delirium and the length of hospital stay when compared to the control group. Nevertheless, no statistically significant differences were observed between the two groups in terms of delirium scores and fall rates. CONCLUSION: In this meta-analysis, HELP can effectively reduce the incidence of delirium and lead to a shorter hospital stay.

Consequences of Preoperative Oral Carbohydrate Consumption in Septal Deviation Patients Undergoing Endoscopic Septoplasty: A Retrospective Cohort Study.

PURPOSE: Multiple reports have demonstrated the benefits of preoperative oral carbohydrates (CHO) in patients receiving open abdominal, thoracic, and orthopedic surgeries. However, thus far, no reports have investigated the benefits of CHO in patients undergoing nasal endoscopic surgery. Our goal was to evaluate the outcome of preoperative oral of administration of CHO in septal deviation patients, undergoing endoscopic septoplasty, under general anesthesia. DESIGN: A retrospective cohort study from a prospectively collected database. METHODS: Consecutive 400 septal deviation patients, undergoing endoscopic septoplasty, were randomly assigned to receive CHO or plain water (80 CHO cohort vs. 320 control cohort) before general anesthesia. The primary outcome was the risk of acute postoperative hypertension (APH). The secondary outcomes included length of hospital stay (LOS), hospitalization cost, sleep time the day before surgery, fluid infusion volume on surgical day, as well the incidence of postoperative nausea and vomiting (PONV) and aspiration. FINDINGS: Patients in the CHO cohort experienced a lower risk of both diastolic blood pressure (DBP)-based APH (OR, 0.49; 95% CI, 0.25 to 0.96; P = 0.0375) and total APH (OR, 0.49; 95% CI, 0.26 to 0.92; P = 0.0258), lower LOS, lower hospitalization cost, longer sleep time and less fluid infusion volume after adjusting for gender, age, BMI, preoperative blood pressure and pulse. Besides, data showed no significant differences in the incidence of (P = 0.4173) and aspiration (P > 0.99). CONCLUSIONS: Preoperative CHO administration can reduce APH risk in patients undergoing endoscopic septoplasty under general anesthesia. Besides, preoperative CHO administration can improve other clinical outcomes, such as, LOS, hospitalization cost, sleep time, and fluid infusion volume. Moreover, CHO safety was confirmed in our study. In the future, additional investigation is necessary to confirm our results.

Structural Basis for Pore Blockade of the Human Cardiac Sodium Channel Nav 1.5 by the Antiarrhythmic Drug Quinidine*.

Nav 1.5, the primary voltage-gated Na+ (Nav ) channel in heart, is a major target for class I antiarrhythmic agents. Here we present the cryo-EM structure of full-length human Nav 1.5 bound to quinidine, a class Ia antiarrhythmic drug, at 3.3 Šresolution. Quinidine is positioned right beneath the selectivity filter in the pore domain and coordinated by residues from repeats I, III, and IV. Pore blockade by quinidine is achieved through both direct obstruction of the ion permeation path and induced rotation of an invariant Tyr residue that tightens the intracellular gate. Structural comparison with a truncated rat Nav 1.5 in the presence of flecainide, a class Ic agent, reveals distinct binding poses for the two antiarrhythmics within the pore domain. Our work reported here, along with previous studies, reveals the molecular basis for the mechanism of action of class I antiarrhythmic drugs.

Dopamine D2 receptors regulate the action potential threshold by modulating T-type calcium channels in stellate cells of the medial entorhinal cortex.

KEY POINTS: Activation of axonal dopamine D2 receptors (D2Rs) increases action potential (AP) threshold, and thus decreases neuronal excitability in layer II stellate cells of medial entorhinal cortex. Endogenous dopamine release increases the AP threshold of stellate cells by activating D2Rs. Activation of D2Rs shifts the activation curve of T-type Ca2+ channels in a positive direction in a protein kinase A-dependent manner. Immunofluorescence staining reveals the presence of T-type Ca2+ channels and D2Rs in the axon initial segments (AISs). This research makes the pioneering discovery of D2R-induced AP threshold plasticity in AISs of stellate cells. The findings are likely to have significant implications for understanding the cellular processes by which dopamine influences neuronal intrinsic excitability. ABSTRACT: Stellate cells in the medial entorhinal cortex (MEC) are considered to constitute the largest population of grid cells, which provide spatial representation to support animal estimation of location. Although dopaminergic fibres from the ventral tegmental area and substantia nigra pars compacta innervate the majority of the cortex, including the MEC, little is known about how dopamine modulates the function of MEC stellate cells. Because dopamine D2 receptors (D2Rs) are involved in spatial cognition and MEC contains high levels of D2Rs, we investigated how D2R activation modulates the neuronal intrinsic excitability of stellate cells. Electrophysiological recordings, optogenetics and molecular biology experiments were performed to investigate the mechanism in mice. Activation of axonal D2Rs, not dendritic or somatic D2Rs, elevated the action potential (AP) threshold and decreased the intrinsic excitability of stellate cells, which was caused by shifting rightward the activation properties of T-type Ca2+ channels in a D2R-protein kinase A-dependent manner without affecting their steady-state inactivation curve. In support, immunofluorescence assays revealed colocalization of D2Rs and Cav 3.2 calcium channels within the axon initial segment. These findings are likely to have significant implications for understanding the cellular processes by which dopamine influences neuronal excitability and they may also be applicable to other hippocampal and cortical regions as dopaminergic fibres innervate wide brain regions. Taken together, these findings provide a novel cellular mechanism by which D2Rs modulate AP threshold of stellate cells through T-type Ca2+ channels in MEC, indicating that D2Rs of MEC play a vital role in modulating the information processing of stellate cells.

ERG3 potassium channel-mediated suppression of neuronal intrinsic excitability and prevention of seizure generation in mice.

KEY POINTS: ERG3 channels have a high expression level in the central nervous system. Knockdown of ERG3 channels enhances neuronal intrinsic excitability (caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials) in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. The expression of ERG3 protein is reduced in human and mouse hippocampal epileptogenic foci. Knockdown of ERG3 channels in hippocampus enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. The results provide strong evidence that ERG3 channels have a crucial role in the regulation of neuronal intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells and are critically involved in the onset and development of epilepsy. ABSTRACT: The input-output relationship of neuronal networks depends heavily on the intrinsic properties of their neuronal elements. Profound changes in intrinsic properties have been observed in various physiological and pathological processes, such as learning, memory and epilepsy. However, the cellular and molecular mechanisms underlying acquired changes in intrinsic excitability are still not fully understood. Here, we demonstrate that ERG3 channels are critically involved in the regulation of intrinsic excitability in hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Knock-down of ERG3 channels significantly increases neuronal intrinsic excitability, which is mainly caused by decreased fast afterhyperpolarization, shortened delay time to the generation of an action potential and enhanced summation of somatic excitatory postsynaptic potentials. Interestingly, the expression level of ERG3 protein is significantly reduced in human and mouse brain tissues with temporal lobe epilepsy. Moreover, ERG3 channel knockdown in hippocampus significantly enhanced seizure susceptibility, while mice treated with the ERG channel activator NS-1643 were less prone to epileptogenesis. Taken together, our results suggest ERG3 channels play an important role in determining the excitability of hippocampal neurons and dysregulation of these channels may be involved in the generation of epilepsy. ERG3 channels may thus be a novel therapeutic target for the prevention of epilepsy.

A Case Report of Sanguis Draconis for Treating a Patient With Refractory Wound Dehiscence After Breast Cancer Chemotherapy.

Breast cancer is one of the most common female malignant tumors. According to data statistics, the incidence of breast cancer was 7% to 10% for a variety of malignant tumors, being only lower than that of uterine cancer. The methods of treating breast cancer are given priority over operative treatment and combined with chemotherapy and radiotherapy. However, exosmosis of chemotherapeutic drugs is a common complication of chemotherapy. Exosmosis of drugs can stimulate local organs to induce acute inflammatory reaction and necrosis, which finally lead to wound infection and difficulty in healing. In December 2013, a patient with full-thickness wound (an area of 5 × 3 cm) dehiscence at the completion of the second phase of chemotherapy for left breast cancer after radical operation was admitted to our department. Her wound had healed after radical operation. The patient followed an integrative therapy treatment protocol that consisted of an external application of a phytomedicine called Sanguis Draconis and combined with a series of conventional treatments, including 3M Transparent Dressing moist therapy, increase in nutrition, and prevention therapies for infection. The patient's integrative treatment program resulted in complete wound healing, and the successful completion of the late 6 courses of chemotherapy. The article describes the nursing experiences associated with this case study.

Sanguis draconis (Daemonorops draco): a case report of treating a chronic pressure ulcer with tunneling.

Pressure ulcers are a frequently encountered difficulty in clinical nursing care. In cases of pressure ulcers, continued pressure on soft tissue leads to pathological processes in affected tissues that include ischemia and hypoxia, nutritional and metabolic disorders, and degeneration and necrosis. Pressure ulcers are a common clinical complication. In February 2013, our department admitted a patient with Parkinson's disease who suffered from a chronic pressure ulcer with tunneling. This patient was given an integrative therapy treatment protocol that consisted of external applications of a phytomedicine called sanguis draconis, combined with a series of conventional treatments, including local oxygen therapy, custom-built vacuum aspiration, and anti-infection therapies. The patient's integrative treatment program resulted in complete amelioration of the pressure ulceration. The following sections describe the nursing experiences associated with this case study.

A proteomics-based study of the mechanism of oxymatrine to ameliorate hepatic fibrosis in mice.

OBJECTIVE: This study investigated the protective effect of oxymatrine (OMT) on carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice and explored its possible targets and signaling pathways. METHODS: Male BALB/c mice were randomly divided into blank control, model, positive drug (silymarin), and OMT administration groups, respectively, with 10 mice in each group. Hepatic fibrosis was induced in mice using CCl4 and the corresponding drug intervention was given. After the final administration, ultrasonography tests, blood tests, and analysis of liver differential proteins using tandem mass tag labeling and liquid chromatography-mass spectrometry were performed. RESULTS: OMT intervention ameliorated CCl4-induced hepatic fibrosis in mice, significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels, down-regulated the expression of fibrosis factors, such as type IV collagen IV, laminin, type III procollagen III, and alpha-smooth muscle actin, and improved liver function. The results of the proteomic analysis showed that the intervention of OMT significantly down-regulated 130 out of 440 up-regulated proteins and up-regulated 70 out of 294 down-regulated proteins, primarily involving the transient receptor potential (TRP) signaling pathway, the peroxisome proliferator-activated receptors (PPAR) signaling pathway, and the metabolic pathway of arachidonic acid. The main differential proteins involved were Cyp2c37, SCP-2, and Tbxas1. In addition, OMT intervention significantly reversed the expression of sterol carrier protein-2 (SCP2) and upregulated the expression of peroxisome proliferator-activated receptor gamma, Cyp2c37, and transient receptor potential cation channel subfamily V member 1 proteins. CONCLUSION: OMT inhibited the proliferative capacity of hepatic stellate cells, induced apoptotic properties, and suppressed the development of fibrosis by elevating Cyp2c37/TRP signaling axis activity and upregulating PPAR pathway activity by inhibiting SCP2.

A versatile residue numbering scheme for Nav and Cav channels.

Voltage-gated sodium (Nav) and calcium (Cav) channels are responsible for the initiation of electrical signals. They have long been targeted for the treatment of various diseases. The mounting number of cryoelectron microscopy (cryo-EM) structures for diverse subtypes of Nav and Cav channels from multiple organisms necessitates a generic residue numbering system to establish the structure-function relationship and to aid rational drug design or optimization. Here we suggest a structure-based residue numbering scheme, centering around the most conserved residues on each of the functional segments. We elaborate the generic numbers through illustrative examples, focusing on representative drug-binding sites of eukaryotic Nav and Cav channels. We also extend the numbering scheme to compare common disease mutations among different Nav subtypes. Application of the generic residue numbering scheme affords immediate insights into hotspots for pathogenic mutations and critical loci for drug binding and will facilitate drug discovery targeting Nav and Cav channels.

Structural basis for human Cav3.2 inhibition by selective antagonists.

The Cav3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Cav3.2 alone and in complex with four T-type calcium channel selective antagonists with overall resolutions ranging from 2.8 Å to 3.2 Å. The four compounds display two binding poses. ACT-709478 and TTA-A2 both place their cyclopropylphenyl-containing ends in the central cavity to directly obstruct ion flow, meanwhile extending their polar tails into the IV-I fenestration. TTA-P2 and ML218 project their 3,5-dichlorobenzamide groups into the II-III fenestration and place their hydrophobic tails in the cavity to impede ion permeation. The fenestration-penetrating mode immediately affords an explanation for the state-dependent activities of these antagonists. Structure-guided mutational analysis identifies several key residues that determine the T-type preference of these drugs. The structures also suggest the role of an endogenous lipid in stabilizing drug binding in the central cavity.