This study was aimed to establish a method to create a stable planar lipid bilayer membranes (PLBMs), in which large conductance calcium-activated potassium channels (BK) were reconstituted. Using spreading method, PLBMs were prepared by decane lipid fluid consisting of N-weathered mixture of phosphatidylcholine and cholesterol at 3:1 ratio. After successful incorporation of BKchannel into PLBMs, single channel characteristics of BKwere studied by patch clamp method. The results showed that i) the single channel conductance of BKwas (206.8 ± 16.9) pS; ii) the activities of BKchannel were voltage dependent; iii) in the bath solution without Ca, there was almost no BKchannel activities regardless of under hyperpolarization or repolarization conditions; iv) under the condition of +40 mV membrane potential, BKchannels were activated in a Caconcentration dependent manner; v) when [Ca] was increased from 1 μmol/L to 100 μmol/L, both the channel open probability and the average open time were increased, and the average close time was decreased from (32.2 ± 2.8) ms to (2.1 ± 1.8) ms; vi) the reverse potential of the reconstituted BKwas -30 mV when [K] was at 40/140 mmol/L (Cis/Trans). These results suggest that the spreading method could serve as a new method for preparing PLBMs and the reconstituted BKinto PLBMs showed similar electrophysiological characteristics to natural BKchannels, so the PLBMs with incorporated BKcan be used in the studies of pharmacology and dynamics of BKchannel.
Animals ; Calcium ; chemistry ; Electrophysiological Phenomena ; Large-Conductance Calcium-Activated Potassium Channels ; chemistry ; Lipid Bilayers ; chemistry ; Membrane Potentials ; Patch-Clamp Techniques

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

Voltage-gated sodium (Na) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na channels, with Na1.1 and Na1.5 each harboring more than 400 mutations. Na channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca) channel Ca1.1 provides a template for homology-based structural modeling of the evolutionarily related Na channels. In this Resource article, we summarized all the reported disease-related mutations in human Na channels, generated a homologous model of human Na1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na channels, the analysis presented here serves as the base framework for mechanistic investigation of Na channelopathies and for potential structure-based drug discovery.
Animals ; Calcium Channels, L-Type ; chemistry ; genetics ; metabolism ; Channelopathies ; genetics ; metabolism ; Humans ; Mutation ; NAV1.1 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; NAV1.7 Voltage-Gated Sodium Channel ; chemistry ; genetics ; metabolism ; Protein Domains ; Rabbits ; Structure-Activity Relationship

The CatSper channel is known as one of the most important Ca²⁺ channels on the cell membrane of mammalian sperm and plays a key role in the motility, hyperactivation and fertilization function of sperm. The CatSper protein, expressed exclusively in the principal piece of the sperm tail, is composed of CatSper1-4 and 5 auxiliary unitsβ,γ,δ and ε, and has an essential part in the functional and structural domains of Ca²⁺as well as in the spatiotemporal regulation of the P-Tyr protein, sperm hyperactivation, efficient sperm migration in the oviduct, egg penetration, and normal fertility. Recent studies show that functional deficiency of CatSper seriously affects sperm function,and the loss of any one of its 9 subunits may lead to male reproductive dysfunction. This paper outlines recent advances in the studies of the CatSperprotein, focusing on its expression, location, structure, and regulation,as well as itsinfluence on sperm hyperactivation and male reproduction.
Animals ; Calcium Channels ; chemistry ; physiology ; Humans ; Infertility, Male ; etiology ; Male ; Sperm Motility ; physiology ; Sperm Tail ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; physiology

OBJECTIVETo review the recent developments in the mechanisms of epithelium sodium channels (ENaCs) induced bone formation and regulation.

DATA SOURCESStudies written in English or Chinese were searched using Medline, PubMed and the index of Chinese-language literature with time restriction from 2005 to 2014. Keywords included ENaC, bone, bone formation, osteonecrosis, estrogen, and osteoporosis. Data from published articles about the structure of ENaC, mechanism of ENaC in bone formation in recent domestic and foreign literature were selected.

STUDY SELECTIONAbstract and full text of all studies were required to obtain. Studies those were not accessible and those did not focus on the keywords were excluded.

RESULTSENaCs are tripolymer ion channels which are assembled from homologous α, β, and γ subunits. Crystal structure of ENaCs suggests that ENaC has a central ion-channel located in the central symmetry axis of the three subunits. ENaCs are protease sensitive channels whose iron-channel activity is regulated by the proteolytic reaction. Channel opening probability of ENaCs is regulated by proteinases, mechanical force, and shear stress. Several molecules are involved in regulation of ENaCs in bone formation, including nitride oxide synthases, voltage-sensitive calcium channels, and cyclooxygenase-2.

CONCLUSIONThe pathway of ENaC involved in shear stress has an effect on stimulating osteoblasts even bone formation by estrogen interference.

Calcium Channels ; physiology ; Epithelial Sodium Channels ; chemistry ; physiology ; Estrogens ; pharmacology ; Humans ; Osteogenesis ; physiology

OBJECTIVETo investigate the protective effects of tea polyphenols (TP) against myocardial ischemia/reperfusion (IR) injuries and explore the possible mechanisms.

METHODSLangendorff-perfused rat hearts were subjected to ischemia for 30 min followed by reperfusion for another 30 min. Myocardial function indices were measured by a left ventricular cannula via a pressure transducer connected to the polygraph in isolated Langendorff hearts and energy metabolism was measured using (31)P nuclear magnetic resonance (NMR) spectroscopy. Whole-cell atch-clamp technique was used to record calcium inward current (I(Ca-L)) in cultured rat cardiac myocytes.

RESULTSCompared with the control hearts, the ex vivo rat hearts with 2.5 mg/L TP treatment showed significantly increased left ventricular developed pressure (LVDP), maximal rise rate of LVDP (+dp/d(tmax)), maximal fall rate of LVDP (-dp/dt(max)), and coronary flow (CF) (P<0.05). During both cardiac ischemia and reperfusion phase, ATP and PCr levels were elevated significantly in TP-treated hearts compared with those in the control hearts (P<0.05). In cultured rat cardiac myocytes, ICa-L was remarkably decreased by TP at the doses of 2.5 and 5.0 mg/L (P<0.01).

CONCLUSIONOur results support a possible protective role of TP against myocardial IR injury by improving myocardial energy metabolism and inhibiting I(Ca-L) in the cardiac myocytes.

Animals ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cells, Cultured ; Energy Metabolism ; Heart ; drug effects ; In Vitro Techniques ; Myocardial Reperfusion Injury ; Myocardium ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Polyphenols ; pharmacology ; Rats ; Tea ; chemistry

TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.
Calcium ; metabolism ; Calcium Channels ; genetics ; metabolism ; Catechols ; pharmacology ; Cell Line ; Fatty Alcohols ; pharmacology ; Gastrointestinal Tract ; drug effects ; metabolism ; Ginger ; chemistry ; Humans ; Nerve Tissue Proteins ; genetics ; metabolism ; Plant Extracts ; pharmacology ; TRPA1 Cation Channel ; Transient Receptor Potential Channels ; genetics ; metabolism

Small conductance Ca(2+)-activated potassium channels (SK channels) distributing in the nervous system play an important role in learning, memory and synaptic plasticity. Most pharmacological properties of them are determined by short-chain scorpion toxins. Different from most voltage-gated potassium channels and large-conductance Ca(2+)-activated potassium channels, SK channels are only inhibited by a small quantity of scorpion toxins. Recently, a novel peptide screener in the extracellular pore entryway of SK channels was considered as the structural basis of toxin selective recognition. In this review, we summarized the unique interactions between scorpion toxins and SK channels, which is crucial not only in deep-researching for physiological function of SK channels, but also in developing drugs for SK channel-related diseases.
Animals ; Memory ; Neuronal Plasticity ; Scorpion Venoms ; chemistry ; Scorpions ; Small-Conductance Calcium-Activated Potassium Channels ; antagonists & inhibitors

This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible.
Animals ; Antihypertensive Agents ; chemistry ; pharmacology ; Calcium Channel Blockers ; chemistry ; pharmacology ; Calcium Channels, L-Type ; genetics ; metabolism ; Drug Repositioning ; methods ; Molecular Structure ; Myocardium ; metabolism ; Rabbits

To build the Dendrobium nobile -T2DM network, and elucidate the molecular mechanism of D. nobile to type 2 diabetes (T2DM). Collect the chemical composition of D. nobile and the targets on T2DM by retrieving database and documents, build the network of D. nobile to T2DM using the entity grammar systems inference rules. The molecular mechanism of D. nobile to T2DM includes: (1) regulating lipid metabolism by lowering triglyceride; (2) reducing insulin resistance; (3) protecting islet cells; (4) promoting the glucose-dependent insulin tropic peptide (GIP) secretion; (5) inhibiting calcium channel. Under the guidance of network pharmacology, through entity grammar systems inference rules we elucidate the molecular mechanism of D. nobile to T2DM, and provide the basis for the further development of health care products based on D. nobile.
Animals ; Calcium Channels ; genetics ; metabolism ; Databases, Factual ; Dendrobium ; chemistry ; Diabetes Mellitus, Type 2 ; drug therapy ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Gene Regulatory Networks ; drug effects ; Humans ; Hypoglycemic Agents ; administration & dosage ; chemistry ; Insulin Resistance ; Islets of Langerhans ; metabolism ; Triglycerides ; metabolism