scholarly journals Functional Cloning Using a Xenopus Oocyte Expression System

10.3791/53518 ◽  
2016 ◽  
Author(s):  
Carol Zygar Plautz ◽  
Hannah C. Williams ◽  
Robert M. Grainger
Keyword(s):  
Expression System ◽  
Oocyte Expression ◽  
Oocyte Expression System

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

Ammonium transport and pH regulation by K+-Cl- cotransporters

2003 ◽  
Vol 285 (1) ◽  
pp. F68-F78 ◽  
Author(s):  
Marc J. Bergeron ◽  
Édith Gagnon ◽  
Bernadette Wallendorff ◽  
Jean-Yves Lapointe ◽  
Paul Isenring
Keyword(s):  
Intracellular Ph ◽  
Cell Membranes ◽  
Ph Regulation ◽  
Expression System ◽  
Kinetic Studies ◽  
Ammonium Transport ◽  
Oocyte Expression ◽  
Oocyte Expression System ◽  
Acidic Environments ◽  
Cellular Acidification

The Na+-K+-Cl- cotransporters (NKCCs), which belong to the cation-Cl- cotransporter (CCC) family, are able to translocate [Formula: see text] across cell membranes. In this study, we have used the oocyte expression system to determine whether the K+-Cl- cotransporters (KCCs) can also transport [Formula: see text] and whether they play a role in pH regulation. Our results demonstrate that all of the CCCs examined (NKCC1, NKCC2, KCC1, KCC3, and KCC4) can promote [Formula: see text] translocation, presumably through binding of the ion at the K+ site. Moreover, kinetic studies for both NKCCs and KCCs suggest that [Formula: see text] is an excellent surrogate of Rb+ or K+ and that [Formula: see text] transport and cellular acidification resulting from CCC activity are relevant physiologically. In this study, we have also found that CCCs are strongly and differentially affected by changes in intracellular pH (independently of intracellular [[Formula: see text]]). Indeed, NKCC2, KCC1, KCC2, and KCC3 are inhibited at intracellular pH <7.5, whereas KCC4 is activated. These results indicate that certain CCC isoforms may be specialized to operate in acidic environments. CCC-mediated [Formula: see text] transport could bear great physiological implication given the ubiquitous distribution of these carriers.

scholarly journals Novel Mutations in the TMPRSS3 Gene May Contribute to Taiwanese Patients with Nonsyndromic Hearing Loss

2020 ◽  
Vol 21 (7) ◽  
pp. 2382
Author(s):  
Swee-Hee Wong ◽  
Yung-Chang Yen ◽  
Shuan-Yow Li ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Serine Protease ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Missense Mutations ◽  
Nonsyndromic Hearing Loss ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Transmembrane Serine Protease

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene’s association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

scholarly journals Signal transduction of rodent opioid μ, δ, κ-receptor in Xenopus oocyte expression system.

1994 ◽  
Vol 64 ◽  
pp. 129
Author(s):  
Takeaki Miyamae ◽  
Nobuyuki Fukushima ◽  
Yoshimi Misu ◽  
Hiroshi Takeshima ◽  
Kazuhiko Fukuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Κ Receptor

Raman Spectroscopic Markers for the Xenopus Laevis Oocyte Expression System

2013 ◽  
Vol 3 (2) ◽  
pp. 151-154 ◽  
Author(s):  
Noriyuki Masuda ◽  
Haim Abitan ◽  
Maria de los Angeles Tejada ◽  
Dan Klaerke ◽  
Henrik Bohr ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Expression System ◽  
Xenopus Laevis Oocyte ◽  
Raman Spectroscopic ◽  
Oocyte Expression ◽  
Oocyte Expression System

cDNA eloping of bovine substance-K receptor through oocyte expression system

Nature ◽  
1987 ◽  
Vol 329 (6142) ◽  
pp. 836-838 ◽  
Author(s):  
Yasuo Masu ◽  
Kazuhisa Nakayama ◽  
Hiromi Tamaki ◽  
Yoshio Harada ◽  
Motoy Kuno ◽  
...  
Keyword(s):  
Expression System ◽  
Oocyte Expression ◽  
Substance K ◽  
Oocyte Expression System
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