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

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

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

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

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.
Animals ; Calcium ; metabolism ; Cattle ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide-Gated Cation Channels ; chemistry ; genetics ; metabolism ; GRB10 Adaptor Protein ; genetics ; metabolism ; Humans ; Kidney ; cytology ; metabolism ; Models, Molecular ; Protein Conformation ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Static Electricity

OBJECTIVETo explore the effect of the total flavonoids of Jiawei Wuzi Yanzong prescription on the Voltage-gated calcium channel of the CA1 pyramidal cell of rat hippocampus.

METHODThe inward calcium current was recorded by the whole-cell patch clamp and the amplitude of it was thought to observe the effect of Abeta25-35 and the total flavonoids. The hippocampus of rat was separated and cut into slices. Active pyramidal cells of slices were chosen for the whole-cell patch clamp recording. After exposure to Abeta25-35, voltage steps (500 ms) were used to depolarize stepwise in a range from 50 to +50 mV (increment: 5 mV). An inward Ca2+ current which was suggested to be survey was evoked. Application of the total flavonoids was to be observed if it had effect on this voltage-depended inward current.

RESULTAbeta25-35 could enhance the calcium current to induce intracellular calcium overload. The amplitude of the control group was--(157.1 +/- 19.9) pA, but after application of Abeta25-35 the current enhanced to--(323.2 +/- 23.4) pA. When the total flavonoids at concentration of 125, 250, 500 g x L(-1) were added, the current declined to--(257.9 +/- 31.6), - (196.4 +/- 29.8) and--(169.3 +/- 34.0) pA, respectively.

CONCLUSIONAbeta-induced intracellular calcium overload may be one of neurotoxic of beta-amyloid peptide. The total flavonoids of Jiawei Wuzi Yanzong prescription can suppress inward calcium current to protect neurons.

Alzheimer Disease ; drug therapy ; metabolism ; physiopathology ; Amyloid beta-Peptides ; metabolism ; Animals ; CA1 Region, Hippocampal ; cytology ; drug effects ; physiology ; Calcium Channels ; genetics ; physiology ; Disease Models, Animal ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Electrophysiology ; Female ; Flavonoids ; chemistry ; pharmacology ; Humans ; Male ; Peptide Fragments ; metabolism ; Prescription Drugs ; chemistry ; pharmacology ; Pyramidal Cells ; drug effects ; physiology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the common rule hepatic energy metabolism on rats by six cold property of traditional Chinese medicines (TCM).

METHODThe activities of Na+ - K+ - ATPase, Ca2+ - ATPase and SDH, the content of hepatic glycogen, and the mRNA expression of hepatic uncoupling protein 2 (UCP2) were measured after the rats and been administrated with water extracts of Radix Sophorae Flavescentis, Fructus Gardeniae, Cortex Phellodendri, Radix Scutellariae, Rhizoma Coptidis, Radix Gentianae respectively at 6.0, 7.0, 8.4, 6.0, 7.0, 4.0 g x kg(-1) doses for 30 days, twice a day.

RESULTThe activities of Na+ - K+ - ATPase, Ca2+ - ATPase and SDH were depressed significantly by six cold property TCM. The decreased tendency of SDH was observed by six cold property of TCM, while statistical significance was obtained in Cortex Phellodendri, Radix Scutellariae, Rhizoma Coptidis, Radix Gentianae. The increased tendency of hepatic glycogen content was found by six cold property TCM, while statistical significance was not obtained in six groups. The decreased tendency of the mRNA expression of UCP2 was found by six cold property TCM, while statistical significance was obtained in Radix Sophorae Flavescentis, Cortex Phellodendri, and Radix Scutellariae groups.

CONCLUSIONThe six cold property TCM have the good effects on hepatic energy metabolism by decreasing the activity of mitochondria SDH to reduce the production of ATP by decreasing the activities of Na+ - K+ - ATPase, Ca2+ - ATPase to cut down the consumption of ATP, by decreasing the mRNA expression of hepatic UCP2 to decrease the heat production.

Adenosine Triphosphate ; metabolism ; Animals ; Calcium-Transporting ATPases ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Energy Metabolism ; drug effects ; Gardenia ; chemistry ; Gentianaceae ; chemistry ; Ion Channels ; genetics ; Liver ; drug effects ; metabolism ; Male ; Mitochondrial Proteins ; genetics ; Polymerase Chain Reaction ; Rats ; Rats, Sprague-Dawley ; Scutellaria baicalensis ; chemistry ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Succinate Dehydrogenase ; metabolism ; Uncoupling Protein 2

OBJECTIVETo establish and optimize a real time RT-PCR system for determining the transcript levels of CatSper1 in human and mouse mature spermatozoa containing microamount of RNA.

METHODSTotal RNA of human and mouse mature spermatozoa was isolated by using TRIzol reagent and reversely transcribed to complementary DNA respectively. Primers for real time RT-PCR were designed in the homologous area of the human and mouse CatSper1 mRNAs. Human sperm complementary DNA was used as the template to the optimize the conditions for SYBR Green I real time RT-PCR, including annealing temperature, Mg2+ concentration, fluorescence measurement temperature and the ratio between forward and reverse primers. The standard curve was constructed with serial dilutions of complementary DNA from human sperm to ascertain the amplification efficiency of SYBR Green I real time PCR and to quantitate the CatSper1 mRNA levels in the human and mouse mature spermatozoa.

RESULTSThe optimal conditions for real time RT-PCR, that is, annealing temperature, Mg2+ concentration and the ratio between forward and reverse primers were 63 degrees C, 3.0 mmol/L and 1:1 respectively. The fluorescence measurement temperature was 88 degrees C. The standard curves were Y = -3.402 log (X) + 25.99 and Y = -3.409 log(X) + 24.09 in the human sperm cDNA and mouse sperm cDNA as the template, with amplification efficiency of 96.8% and 96.5% respectively. The R2 value (an indicator of the quality of the fit of the standard curve to the standard data points plotted) of both standard curves was 0.998. The CatSper1 mRNA levels in the human and mouse mature spermatozoa could be determined according to the standard curve.

CONCLUSIONThe general RT-PCR system, by adding SYBR Green I and optimizing its conditions, could be used to quantitate the mRNA levels in both human and mouse mature spermatozoa.

Animals ; Calcium Channels ; genetics ; Humans ; Male ; Mice ; Organic Chemicals ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Spermatozoa ; metabolism

Hypokalemic periodic paralysis (HOPP) is a rare disease characterized by reversible attacks of muscle weakness accompanied by episodic hypokalemia. Recent molecular work has revealed that the majority of familial HOPP is due to mutations in a skeletal muscle voltage-dependent calcium-channel: the dihydropyridine receptor. We report a 13-yr old boy with HOPP from a family in which 6 members are affected in three generations. Genetic examination identified a nucleotide 3705 C to G mutation in exon 30 of the calcium channel gene, CACNA1S. This mutation predicts a codon change from arginine to glycine at the amino acid position #1239 (R1239G). Among the three known mutations of the CACNA1S gene, the R1239G mutation was rarely reported. This boy and the other family members who did not respond to acetazolamide, showed a marked improvement of the paralytic symptoms after spironolactone treatment.
Acetazolamide/pharmacology ; Adolescent ; Arginine/chemistry ; Calcium Channels/chemistry/*genetics ; Codon ; Exons ; Family Health ; Female ; Glycine/chemistry ; Humans ; Hypokalemia/metabolism ; Hypokalemic Periodic Paralysis/*diagnosis/*genetics ; Korea ; Male ; Muscle, Skeletal/metabolism ; Mutation ; Pedigree ; Protein Structure, Tertiary ; Sequence Analysis, DNA ; Spironolactone/pharmacology