Animals ; Oocytes ; metabolism ; physiology ; Receptors, G-Protein-Coupled ; physiology ; Receptors, Purinergic P2X ; physiology ; Xenopus laevis

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.
Animals ; Artificial Intelligence ; Cryoelectron Microscopy ; Nuclear Pore/ultrastructure* ; Oocytes/metabolism* ; Xenopus laevis

The antimicrobial peptide magainin II is expressed in the skin of the African clawed frog, Xenopus laevis, and exhibits a broad spectrum of antimicrobial activity as well as tumoricidal properties at low concentrations. In addition, magaininII plays a synergistic role during antimicrobial and tumoricidal processes with another antimicrobial peptide PGLa that is also expressed in Xenopus laevis. The optimized cDNA sequence of magainin II and magainin II-PGLa hybrid peptide according to E. coli or Pichia pastoris codon usage frequency were synthesized and sub-cloned into prokaryotic expression vector pGEX and Pichia pastoris secreted expression vector pPIC9k. The resulting recombinant plasmids were named as pGEX-magainin II and pPIC9k-magainin II-PGLa. The GST-magainin II fusion protein was highly expressed in E. coli. Furthermore, magainin II was successfully purified by digestion with PreScission Protease to cleave the GST tag. Additionally, our data obtained from the ELISA revealed that magainin II -PGLa hybrid peptide was successfully expressed in Pichia pastoris. These experiments establish a useful system for further studies of these antimicrobial peptides.
Animals ; Escherichia coli ; metabolism ; Genetic Vectors ; Magainins ; biosynthesis ; genetics ; Peptides ; genetics ; metabolism ; Pichia ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Xenopus Proteins ; biosynthesis ; genetics ; Xenopus laevis

AIMTo explore a method of the stable and persistent expression of HERG(human ether-a-go-go-related gene) channels in Xenopus oocytes, and investigate the alteration of rest membrane potential of oocytes and electrophysiological properties of expressed channel in different culture duration.

METHODSHERG mRNA for injection was prepared with in intro transcription using vector plasmid pSP64 containing HERG cDNA fragment. Expressed HERG current was recorded using standard two-microelectrode voltage-clamp technique.

RESULTS(1) Functional channels, with electrophysiological properties consistent with those of HERG channels were persistently expressed in oocytes membrane with this method. Furthermore, channel current could be recorded stably in 10-15 days. (2) The negative value of rest membrane potential increased gradually in the 3, 6, and 9 days of culture, and then decreased in the 12 days. The potential of peak value of inward rectification shifted gradually to the positive direction in 3, 6 and 9 days, and recovered in 12 days. Half-maximal activation potential (V1/2) of heterological expressed current shifted gradually to the negative direction in 3, 6 and 9 days of culture and then recovered in 12 days, the tendency of change was coincident with that of membrane rest potential.

CONCLUSIONThe investigation provides a method of persistent expression of HERG channel in Xenopus oocytes and offers evidences for the difference of electrophysiological experimental data of studies of molecular site and drugs effect of HERG channel in different experimental conditions.

Animals ; Ether-A-Go-Go Potassium Channels ; genetics ; metabolism ; Humans ; Membrane Potentials ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Xenopus laevis

OBJECTIVETo investigate the characteristic and effect of cadmium on ATP-activated currents (I(ATP)) mediated by P2X4 purinoceptors.

METHODSTranscribe cDNA coding for the rat P2X4 receptor to cRNA in vitro. Inject the cRNA to oocytes of an xenopus laevis using the microinjection technique. Reveal the effect of cadmium on I(ATP) mediated by P2X4 receptor using the two-electrode whole-cell voltage clamp technique.

RESULTS(1) Within a certain concentration range, cadmium was found to reversibly magnify I(ATP) mediated by P2X4 receptors expressed in oocytes of an xenopus. When the concentration of cadmium reached 30 micromol/L, the increase of I(ATP) was the most significant. I(ATP) turned to decrease when the concentration of cadmium was more than 30 micromol/L; (2) The concentration-response curve was shifted to left by applying cadmium at 10 micromol/L; the EC50 was reduced from (17.1 +/- 1.5) micromol/L to (9.8 +/- 1.8) micromol/L (n = 6, P < 0.01) and the Hill coefficient was increased from 1.14 +/- 0.13 to 1.57 +/- 0.36; (3) The effect of cadmium on I(ATP) showed no dependence on membrane voltage; (4) The magnifying effect on I(ATP) reached maximum when preincubating cadmium for 120 seconds.

CONCLUSIONThe increase I(ATP) by cadmium is reversible, concentration-dependent, time-dependent, and voltage-independent. One reason of this augment effect could be the allosteric modulation on P2X4 receptors.

Adenosine Triphosphate ; metabolism ; Animals ; Cadmium ; toxicity ; Microinjections ; Oocytes ; drug effects ; metabolism ; physiology ; Rats ; Receptors, Purinergic P2X4 ; metabolism ; Xenopus laevis

AIMTo determine the feasibility of establishing the heterologous expression model of human- serotonin transporter(hSERT or 5-HTT).

METHODScRNA of SERT was transcribed from cDNA, which was cloned in the pOTV vector. Each oocyte of mature xenopus laevis was injected with transcribed cRNA in vivo and incubated at room temperature for 4-9 days. Recording the current induced by 5-HT with voltage clamp technique tested the function of the expressed 5-HT transporter.

RESULTSThe transporter current could be observed in Ringer's solution containing 5-HT, and the 5-HT induced current were concentration-dependent. Norepinephrine and dopamine could not induce the transporter current while the 5-HT induced current could be specifically inhibited by 5-HTT blocker, desipramine.

CONCLUSIONThe results demonstrate that the heterologous expression product in xenopus laevis oocytes is human 5-HT transporter.

Animals ; Carrier Proteins ; genetics ; DNA, Complementary ; genetics ; Female ; Gene Expression ; Models, Animal ; Oocytes ; metabolism ; RNA, Messenger ; genetics ; Serotonin ; metabolism ; Serotonin Plasma Membrane Transport Proteins ; biosynthesis ; genetics ; Xenopus laevis

We have investigated the methods and mechanisms for analysis of the channel kinetics parameters of voltage-gated potassium channels, HERG (Human ether-à-go-go related gene) channels, in the process of electrophysiological recording. The current of HERG K+ channels expressed in Xenopus oocytes was studied using a two-electrode voltage clamp technique, and the channel kinetics parameters were analyzed through compiling different pulse protocol and recording the current. Results showed: (1) The HERG K+ channels, under conditions of being activated with depolarized pulse, expressed an inward-rectified property attributing to rapid inactivation. The activation curve could be obtained through fitting the depolarized potential and the following peak amplitude of tail current, while the parameters of time-dependent activation was obtained through fitting different depolarized duration and the corresponding peak amplitude of tail current. (2) The I-V relationship still exhibit marked inward rectification. Tail current decay traces were fitted with a bi-exponential function to determine the time constants of the fast and slow components of current decay. (3) The inactivation of HERG channels is voltage-dependent. The inactivation process was isolated with two different three-pulse protocols, with which the inactivation curve and nearly linear I-V relationship were obtained, respectively. Thus, altough the kinetics properties of HERG channels were complicated, the channels kinetics could be indirectly analyzed through differently designed pulse protocols, which provided the basis for investigation on Alanine-scanning mutagenesis and agent action.
Animals ; ERG1 Potassium Channel ; Ether-A-Go-Go Potassium Channels ; analysis ; genetics ; Humans ; Kinetics ; Oocytes ; metabolism ; Patch-Clamp Techniques ; Xenopus laevis ; metabolism

BACKGROUNDKetanserin (KT), a selective serotonin (5-HT) 2-receptor antagonist, reduces peripheral blood pressure by blocking the activation of peripheral 5-HT receptors. In this study electrophysiological method was used to investigate the effect of KT and potassium ion on Kv1.3 potassium channels and explore the role of blocker KT in the alteration of channel kinetics contributing to the potassium ion imbalances.

METHODSKv1.3 channels were expressed in xenopus oocytes, and currents were measured using the two-microelectrode voltage-clamp technique.

RESULTSKCl made a left shift of activation and an inactivation curve of Kv1.3 current and accelerated the activation and inactivation time constant. High extracellular [K(+)] attenuated the blockade effect of KT on Kv1.3 channels. In the presence of KT and KCl the activation and inactivation time constants were not influenced significantly no matter what was administered first. KT did not significantly inhibit Kv1.3 current induced by tetraethylammonium (TEA).

CONCLUSIONSKT is a weak blocker of Kv1.3 channels at different concentrations of extracellular potassium and binds to the intracellular side of the channel pore. The inhibitor KT of ion channels is not fully effective in clinical use because of high [K(+)](o) and other electrolyte disorders.

Animals ; Electrophysiology ; Female ; Ketanserin ; pharmacology ; Kv1.3 Potassium Channel ; drug effects ; metabolism ; Oocytes ; Patch-Clamp Techniques ; Potassium ; pharmacology ; Serotonin Antagonists ; pharmacology ; Xenopus laevis

OBJECTIVETo investigate the molecular mechanism of curcumin in reducing blood lipids by establishing gene expression system of human low density lipoprotein receptors (LDL-R) in Xenopus Laevis oocytes (XLO).

METHODSThe expression of LDL-R on cytomembrane was determined using immuno-fluorescent, ligand-fluorescent and immune colloidal gold techniques after human LDL-R containing p3.7 LDL plasmid was led into nucleus. And the expression of LDL-R gene in XLO was quantitatively determined by ELISA after being interfered with different concentrations of curcumin.

RESULTSThe human LDL-R gene could be expressed on XLO, which could be significantly enhanced by curcumin in a dose-dependent manner. Conclusion One of the paths of curcumin in reducing blood lipids and anti-atherosclerosis was improving LDL-R gene expression and increasing the LDL-cholesterol absorption of cells.

Animals ; Cells, Cultured ; Curcumin ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Gene Expression Regulation ; Humans ; Microinjections ; Oocytes ; cytology ; metabolism ; Receptors, LDL ; biosynthesis ; genetics ; Xenopus laevis

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