scholarly journals Heterologous Expression of Aedes aegypti Cation Chloride Cotransporter 2 (aeCCC2) in Xenopus laevis Oocytes Induces an Enigmatic Na+/Li+ Conductance

Insects ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 71 ◽  
Author(s):  
Megha Kalsi ◽  
Christopher Gillen ◽  
Peter Piermarini
Keyword(s):  
Plasma Membrane ◽  
Aedes Aegypti ◽  
Heterologous Expression ◽  
Xenopus Oocytes ◽  
Xenopus Laevis Oocytes ◽  
Conductive Pathway ◽  
Electrode Voltage ◽  
Genes Encoding ◽  
Voltage Clamping ◽  
Cation Chloride Cotransporters

The yellow fever mosquito Aedes aegypti possesses three genes encoding putative Na+-coupled cation chloride cotransporters (CCCs): aeNKCC1, aeCCC2, and aeCCC3. To date, none of the aeCCCs have been functionally characterized. Here we expressed aeCCC2 heterologously in Xenopus oocytes and measured the uptake of Li+ (a tracer for Na+) and Rb+ (a tracer for K+). Compared to control (H2O-injected) oocytes, the aeCCC2-expressing oocytes exhibited significantly greater uptake of Li+, but not Rb+. However, the uptake of Li+ was neither Cl−-dependent nor inhibited by thiazide, loop diuretics, or amiloride, suggesting unconventional CCC activity. To determine if the Li+-uptake was mediated by a conductive pathway, we performed two-electrode voltage clamping (TEVC) on the oocytes. The aeCCC2 oocytes were characterized by an enhanced conductance for Li+ and Na+, but not K+, compared to control oocytes. It remains to be determined whether aeCCC2 directly mediates the Na+/Li+ conductance or whether heterologous expression of aeCCC2 stimulates an endogenous cation channel in the oocyte plasma membrane.

Inhibitory Effect of Glucocorticoids on Human-cloned 5-hydroxytryptamine3AReceptor Expressed in Xenopus  Oocytes

2004 ◽  
Vol 101 (3) ◽  
pp. 660-665 ◽  
Author(s):  
Takahiro Suzuki ◽  
Masahiro Sugimoto ◽  
Hideki Koyama ◽  
Takashi Mashimo ◽  
Ichiro Uchida
Keyword(s):  
Xenopus Oocytes ◽  
Inhibitory Concentration ◽  
Inhibitory Effect ◽  
Combined Effect ◽  
Induced Current ◽  
Direct Effects ◽  
Receptor Antagonists ◽  
Electrode Voltage ◽  
Direct Inhibitory Effect ◽  
Voltage Clamping

Background Methylprednisolone, dexamethasone, and other glucocorticoids have been found effective against nausea and vomiting induced by chemotherapy and surgery. Although the specific 5-hydroxytriptamine3 (5-HT3) receptor antagonists such as ondansetron and ramosetron are used as antiemetics, reports show that the use of 5-HT3 receptor antagonists with some glucocorticoids brings additional effects. Glucocorticoids are reported to be antiemetic. The effect of glucocorticoids on 5-HT3 receptor, however, has not been well characterized. This study was designed to examine whether dexamethasone and methylprednisolone had direct effects on human-cloned 5-HT3A receptor expressed in Xenopus oocytes. Methods Homomeric human-cloned 5-HT3A receptor was expressed in Xenopus oocytes. The authors used the two-electrode voltage-clamping technique to study the effect of methylprednisolone and dexamethasone on 5-HT-induced current. Results Both dexamethasone and methylprednisolone concentration-dependently attenuated 5-HT-induced current. Dexamethasone inhibited 2 microm 5-HT-induced current, which was equivalent to EC30 concentration for 5-HT3A receptor, with an inhibitory concentration 50% of 5.29 +/- 1.02 microm. Methylprednisolone inhibited 2 microm 5-HT-induced current with an inhibitory concentration 50% of 1.07 +/- 0.15 mm. The mode of inhibition with either dexamethasone or methylprednisolone was noncompetitive and voltage-independent. When administered together with the 5-HT3 receptor antagonists, ramosetron or metoclopramide, both glucocorticoids showed an additive effect on 5-HT3 receptor. Conclusion The glucocorticoids had a direct inhibitory effect on 5-HT3 receptors. The combined effect of glucocorticoids and the 5-HT3 receptor antagonists seems additive.

scholarly journals Effects of tranexamic acid on the activity of glutamate transporter EAAT3

2020 ◽  
Vol 15 (3) ◽  
pp. 291-296
Author(s):  
Hyun-Jung Shin ◽  
Soo-Young Lee ◽  
Hyo-Seok Na ◽  
Bon-Wook Koo ◽  
Jung-Hee Ryu ◽  
...  
Keyword(s):  
Tranexamic Acid ◽  
Excitatory Amino Acid ◽  
Glutamate Transporter ◽  
Control Group ◽  
High Dose ◽  
Xenopus Laevis Oocytes ◽  
Excitatory Amino Acid Transporter ◽  
Electrode Voltage ◽  
Voltage Clamping ◽  
Type 3

Background: Tranexamic acid (TXA) is the most widely used hemostatic agent in surgical patients. However, when used in a high dose, it could cause a seizure in the postoperative period. The exact effector mechanism behind the seizure triggering remains unknown. Therefore, the authors investigated the effects of TXA on the activity of glutamate transporter type 3 (excitatory amino acid transporter 3; EAAT3), which is the main neuronal glutamate transporter type. Methods: EAAT3 was expressed in Xenopus laevis oocytes through mRNA injection. Oocytes were incubated with diluted tranexamic acid for 72 h. Two-electrode voltage clamping was used to measure membrane currents before, during, and after applying 30 M L-glutamate. Responses were quantified by integrating the current traces and reported in microcoulombs (C). Results were presented as mean  SEM.Results: TXA (30 to 1,000 M) significantly decreased EAAT3 activity. Our kinetic study showed that Vmax was significantly decreased in the TXA group compared with the control group (1.1  0.1 vs. 1.4  0.1 C, n = 18–23, P = 0.043), but the Km did not significantly change (12.7  3.9 M for TXA vs. 12.8  3.8 for control, n = 18–23, P = 0.986).Conclusions: Our results suggest that TXA attenuates EAAT3 activity, which may explain its proconvulsant effect.

Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin

1996 ◽  
Vol 109 (6) ◽  
pp. 1285-1295
Author(s):  
F. Le Caherec ◽  
P. Bron ◽  
J.M. Verbavatz ◽  
A. Garret ◽  
G. Morel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Digestive Tract ◽  
Xenopus Oocytes ◽  
Plasma Membranes ◽  
Expression System ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Plasma Membrane Proteins ◽  
Cicadella Viridis

Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 × 10(−3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 × 10(−3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.

scholarly journals NHE8 is an intracellular cation/H+ exchanger in renal tubules of the yellow fever mosquito Aedes aegypti

2009 ◽  
Vol 296 (4) ◽  
pp. F730-F750 ◽  
Author(s):  
Peter M. Piermarini ◽  
Dirk Weihrauch ◽  
Heiko Meyer ◽  
Markus Huss ◽  
Klaus W. Beyenbach
Keyword(s):  
Aedes Aegypti ◽  
Yellow Fever ◽  
Xenopus Oocytes ◽  
Intracellular Localization ◽  
Malpighian Tubules ◽  
Xenopus Laevis Oocytes ◽  
Renal Tubules ◽  
Yellow Fever Mosquito ◽  
Western Blots ◽  
Principal Cells

The goal of this study was to identify and characterize the hypothesized apical cation/H+ exchanger responsible for K+ and/or Na+ secretion in the renal (Malpighian) tubules of the yellow fever mosquito Aedes aegypti. From Aedes Malpighian tubules, we cloned “ AeNHE8,” a full-length cDNA encoding an ortholog of mammalian Na+/H+ exchanger 8 (NHE8). The expression of AeNHE8 transcripts is ubiquitous among mosquito tissues and is not enriched in Malpighian tubules. Western blots of Malpighian tubules suggest that AeNHE8 is expressed primarily as an intracellular protein, which was confirmed by immunohistochemical localizations in Malpighian tubules. AeNHE8 immunoreactivity is expressed in principal cells of the secretory, distal segments, where it localizes to a subapical compartment (e.g., vesicles or endosomes), but not in the apical brush border. Furthermore, feeding mosquitoes a blood meal or treating isolated tubules with dibutyryl-cAMP, both of which stimulate a natriuresis by Malpighian tubules, do not influence the intracellular localization of AeNHE8 in principal cells. When expressed heterologously in Xenopus laevis oocytes, AeNHE8 mediates EIPA-sensitive Na/H exchange, in which Li+ partially and K+ poorly replace Na+. The expression of AeNHE8 in Xenopus oocytes is associated with the development of a conductive pathway that closely resembles the known endogenous nonselective cation conductances of Xenopus oocytes. In conclusion, AeNHE8 does not mediate cation/H+ exchange in the apical membrane of Aedes Malpighian tubules; it is more likely involved with an intracellular function.

Regulation of alpha 1-beta 3-NA(+)-K(+)-ATPase isozyme during meiotic maturation of Xenopus laevis oocytes

1992 ◽  
Vol 262 (6) ◽  
pp. C1520-C1530 ◽  
Author(s):  
D. Pralong-Zamofing ◽  
Q. H. Yi ◽  
G. Schmalzing ◽  
P. Good ◽  
K. Geering
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Binding Capacity ◽  
Active Form ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Cell Fractionation ◽  
Binding Studies ◽  
Ouabain Binding

During progesterone-induced maturation of Xenopus oocytes, the transport and ouabain binding capacity of Na(+)-K(+)-ATPase at the plasma membrane is completely downregulated. To elucidate the mechanism and the physiological significance of this process, we have followed the fate of oocyte alpha-beta 3-Na(+)-K(+)-ATPase complexes during meiotic maturation and early embryonic development. An immunocytochemical follow-up of the catalytic alpha-subunit, ouabain binding studies, cell surface iodination, and oocyte cell fractionation combined with immunochemical subunit detection provides evidence that following progesterone treatment Na(+)-K(+)-ATPase molecules are retrieved from the oocyte plasma membrane. The enzyme complexes are recovered in an active form in an intracellular compartment in both in vitro and in vivo matured eggs. Exogenous Xenopus alpha 1- and beta 1-complexes expressed in the oocyte from injected cRNAs are regulated by progesterone similar to endogenous Na(+)-K(+)-ATPase complexes. Finally, active Na(+)-K+ pumps internalized during oocyte maturation appear to be redistributed to plasma membrane fractions during blastula formation in Xenopus embryos. In conclusion, our data suggest that endocytosis of alpha 1- and beta 3-complexes during meiotic maturation of Xenopus oocytes is responsible for downregulation of Na(+)-K(+)-ATPase activity and results in an intracellular pool of functional enzymes, which might be reexpressed during early development in response to physiological needs.

Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes

1994 ◽  
Vol 267 (6) ◽  
pp. C1717-C1722 ◽  
Author(s):  
K. Rezai ◽  
A. Kulisz ◽  
W. J. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Stage Iv ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Cell Systems ◽  
Oocyte Meiotic Maturation

Progesterone-stimulated Xenopus laevis oocytes undergo an increase in their intracellular pH from 7.3 to 7.7 because of the activation of Na+/H+ antiporters in their plasma membrane. Activation of Na+/H+ exchangers (NHE) in other cell systems appears to be regulated by phosphorylation of the NHE protein. In the current study we demonstrated that cytoplasm taken from steroid-stimulated oocytes rapidly induced an increase in intracellular pH when microinjected into full-grown stage VI recipient oocytes. The protein within the cytoplasm that appears to be responsible for this activity is c-mos kinase. Microinjected pure mosxe kinase protein rapidly activated the Na+/H+ exchangers in full-grown recipient oocytes. Furthermore, injected mosxe protein rapidly activated the Na+/H+ exchangers in smaller progesterone-insensitive stage IV oocytes. Therefore, it appears that the protooncogene product, p39 c-mos kinase, which is normally synthesized in full-grown stage VI oocytes in response to progesterone stimulation, is involved in the upregulation of the Na+/H+ antiporters during oocyte meiotic maturation.

Expression of inositol 1,4,5-trisphosphate receptors changes the Ca2+ signal of Xenopus oocytes

1996 ◽  
Vol 270 (4) ◽  
pp. C1255-C1261 ◽  
Author(s):  
S. DeLisle ◽  
O. Blondel ◽  
F. J. Longo ◽  
W. E. Schnabel ◽  
G. I. Bell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Expression Level ◽  
Xenopus Laevis Oocytes ◽  
Biological Functions ◽  
Propagating Waves ◽  
Inositol 1,4,5 Trisphosphate ◽  
Related Proteins ◽  
Type 3

The receptors for the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] form a family of closely related proteins that play an important role in regulating the free intracellular Ca2+ concentration. To test the hypothesis that changing the expression level of Ins(1,4,5)P3 receptors could alter the Ins(1,4,5)P3-mediated Ca2+ signal, we overexpressed Ins(1,4,5)P3 receptor type 1 (InsP3R-1) or type 3 (InsP3R-3) in Xenopus laevis oocytes. Expression of InsP3R-1 increased the velocity of the propagating waves of intracellular Ca2+ release but did not affect the Ins(1,4,5)P3-induced entry of extracellular Ca2+ across the plasma membrane. In contrast, expression of intracellular Ca2+ but markedly increased the magnitude and duration of Ca2+ influx. Immunolocalization studied revealed InsP3R-3 at the endoplasmic reticulum, with a relatively stronger signal at or near the plasma membrane. The results suggest that changing the expression level of an InsP3R can alter the Ins(1,4,5)P3-mediated Ca2+ signal and that InsP3R-1 and InsP3R-3 may have different biological functions.

scholarly journals Electrical recordings of the mitochondrial calcium uniporter in Xenopus oocytes

2018 ◽  
Vol 150 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Chen-Wei Tsai ◽  
Ming-Feng Tsai
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Electrophysiological Recordings ◽  
Electrode Voltage ◽  
Genes Encoding ◽  
Functional Mechanisms ◽  
Inwardly Rectifying

The mitochondrial calcium uniporter is a multisubunit Ca2+ channel that mediates mitochondrial Ca2+ uptake, a cellular process crucial for the regulation of oxidative phosphorylation, intracellular Ca2+ signaling, and apoptosis. In the last few years, genes encoding uniporter proteins have been identified, but a lack of efficient tools for electrophysiological recordings has hindered quantitative analysis required to determine functional mechanisms of this channel complex. Here, we redirected Ca2+-conducting subunits (MCU and EMRE) of the human uniporter to the plasma membrane of Xenopus oocytes. Two-electrode voltage clamp reveals inwardly rectifying Ca2+ currents blocked by a potent inhibitor, Ru360 (half maximal inhibitory concentration, ~4 nM), with a divalent cation conductivity of Ca2+ > Sr2+ > Ba2+, Mn2+, and Mg2+. Patch clamp recordings further reveal macroscopic and single-channel Ca2+ currents sensitive to Ru360. These electrical phenomena were abolished by mutations that perturb MCU-EMRE interactions or disrupt a Ca2+-binding site in the pore. Altogether, this work establishes a robust method that enables deep mechanistic scrutiny of the uniporter using classical strategies in ion channel electrophysiology.

scholarly journals Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

1995 ◽  
Vol 15 (10) ◽  
pp. 5508-5515 ◽  
Author(s):  
M E Drew ◽  
C K Langford ◽  
E M Klamo ◽  
D G Russell ◽  
M P Kavanaugh ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Leishmania Donovani ◽  
External Surface ◽  
Functional Expression ◽  
Xenopus Laevis Oocytes ◽  
Surface Molecules ◽  
Inositol Phospholipids ◽  
Electrode Voltage ◽  
Genus Leishmania ◽  
Specific Polyclonal Antibody

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.

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