Amino Acid Substitution ; Drug Resistance, Multiple, Bacterial ; physiology ; Escherichia coli ; physiology ; Escherichia coli Proteins ; genetics ; metabolism ; Membrane Potentials ; physiology ; Models, Biological ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Mutation, Missense

To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.
Adenosine Triphosphate ; antagonists & inhibitors ; pharmacology ; Animals ; Female ; Magnesium ; pharmacology ; Membrane Potentials ; drug effects ; Oocytes ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Receptors, Purinergic P2X4 ; genetics ; physiology ; Xenopus laevis

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).
Amino Acid Sequence ; Animals ; Antibodies, Blocking ; metabolism ; pharmacology ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; genetics ; immunology ; metabolism ; Guinea Pigs ; Membrane Potentials ; Molecular Sequence Data ; Myocytes, Cardiac ; drug effects ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors ; genetics ; immunology

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
Animals ; Cell Line ; Chloride Channels ; physiology ; Cricetinae ; Cystic Fibrosis Transmembrane Conductance Regulator ; biosynthesis ; genetics ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Membrane Potentials ; genetics ; Protein Processing, Post-Translational ; Protein Splicing

During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.
Animals ; Calcium/metabolism ; Calcium Channels/metabolism ; Calcium Channels, L-Type/genetics/metabolism ; Cations/metabolism ; *Cell Differentiation ; *Cell Proliferation ; Cells, Cultured ; Excitation Contraction Coupling ; Gene Knockdown Techniques ; Membrane Potentials ; Mice ; Muscle Fibers, Skeletal/*metabolism ; Muscle Proteins/metabolism ; Myoblasts, Skeletal/*metabolism ; Ryanodine Receptor Calcium Release Channel/metabolism ; Sarcoplasmic Reticulum/*physiology ; Synaptophysin/metabolism ; TRPC Cation Channels/genetics/*metabolism ; Transient Receptor Potential Channels/metabolism

PURPOSE: This study examined the expression and function of inward rectifier K+ channels in cultured rat hepatic stellate cells (HSC). MATERIALS AND METHODS: The expression of inward rectifier K+ channels was measured using real-time RT-PCR, and electrophysiological properties were determined using the gramicidin-perforated patch-clamp technique. RESULTS: The dominant inward rectifier K+ channel subtypes were K(ir)2.1 and K(ir)6.1. These dominant K+ channel subtypes decreased significantly during the primary culture throughout activation process. HSC can be classified into two subgroups: one with an inward-rectifying K+ current (type 1) and the other without (type 2). The inward current was blocked by Ba2+ (100micrometer) and enhanced by high K+ (140mM), more prominently in type 1 HSC. There was a correlation between the amplitude of the Ba2+-sensitive current and the membrane potential. In addition, Ba2+ (300micrometer) depolarized the membrane potential. After the culture period, the amplitude of the inward current decreased and the membrane potential became depolarized. CONCLUSION: HSC express inward rectifier K+ channels, which physiologically regulate membrane potential and decrease during the activation process. These results will potentially help determine properties of the inward rectifier K+ channels in HSC as well as their roles in the activation process.
Animals ; Barium/pharmacology ; Blotting, Western ; Cells, Cultured ; Electrophysiology ; Liver/cytology/*metabolism ; Male ; Membrane Potentials/drug effects ; Potassium/pharmacology ; Potassium Channels, Inwardly Rectifying/genetics/metabolism/*physiology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

Action Potentials ; Animals ; Female ; Heart ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Knockout ; Myocardium ; metabolism ; Patch-Clamp Techniques ; Sodium Channels ; physiology

OBJECTIVETo investigate the ways testosterone influences the murine bone marrow-derived macrophages (BMMs) and how testosterone affects the function of BMMs after bound to their membrane surface.

METHODSBMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intracellular androgen receptor (AR) detected through RT-PCR and Western blotting. The binding site of testosterone (T) to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca2+ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp.

RESULTSRT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca2+ through Ni2+ -blockable Ca2+ channels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs.

CONCLUSIONIt is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca2+ influx and a rapid rise in the intracellular Ca2+ concentration, and influence the function of BMMs.

Animals ; Blotting, Western ; Calcium ; metabolism ; Calcium Channels ; physiology ; Cell Membrane ; metabolism ; Cells, Cultured ; Female ; Macrophages ; cytology ; metabolism ; physiology ; Membrane Potentials ; Mice ; Mice, Inbred C57BL ; Microscopy, Confocal ; Protein Binding ; Receptors, Androgen ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Testosterone ; metabolism

Because HERG potassium channel has important effects on both proarrhythmia and antiarrhythmia, we use immunofluorescence and Western blotting methods to detect the expression of HERG channel of HERG-HEK cells in different concentrations of matrine, oxymatrine and resveratrol. The findings showed that both matrine (1 micromol x L(-1) ) and oxymatrine ( 1micromol x L (-1) ) increased HERG channel expression ( n = 5, P < 0. 05 ) , while matrine (100 micromol x L(-1) ) decreased HERG channel expression ( n = 5, P < 0. 05), resveratrol didn't affect HERG channel expression. In conclusion, different concentrations of matrine and oxymatrine affect HERG channel expression, while there is no relationship between resveratrol and HERG channel expression. It provides a theoretical support for the safety and mechanism of anti-arrhythmic drugs.
Alkaloids ; pharmacology ; Anti-Arrhythmia Agents ; pharmacology ; Blotting, Western ; Cell Line ; Dose-Response Relationship, Drug ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; genetics ; metabolism ; physiology ; Fluorescent Antibody Technique ; Humans ; Membrane Potentials ; drug effects ; Patch-Clamp Techniques ; Plants, Medicinal ; chemistry ; Quinolizines ; pharmacology ; Sophora ; chemistry ; Stilbenes ; pharmacology