Biophysical Approach to Determine the Subunit Stoichiometry of the Epithelial Sodium Channel Using the Xenopus laevis Oocyte Expression System

Ion Channels ◽  
2006 ◽  
pp. 53-63
Author(s):  
Farhad Kosari ◽  
Shaohu Sheng ◽  
Thomas R. Kleyman
Keyword(s):  
Xenopus Laevis ◽  
Sodium Channel ◽  
Expression System ◽  
Epithelial Sodium Channel ◽  
Xenopus Laevis Oocyte ◽  
Subunit Stoichiometry ◽  
Oocyte Expression ◽  
Biophysical Approach ◽  
Oocyte Expression System

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

Drug transport studies by human MDR1 in Xenopus laevis oocyte expression system

2008 ◽  
Vol 22 (S2) ◽  
pp. 633-633
Author(s):  
Promsuk Jutabha ◽  
Shinichi Narikawa ◽  
Yi Xiu‐Lin ◽  
Naoko Ohtsu ◽  
Jun Otomo ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Drug Transport ◽  
Expression System ◽  
Xenopus Laevis Oocyte ◽  
Transport Studies ◽  
Oocyte Expression ◽  
Oocyte Expression System ◽  
Human Mdr1

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.

scholarly journals Cellular encoding of Cy dyes for single-molecule imaging

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lilia Leisle ◽  
Rahul Chadda ◽  
John D Lueck ◽  
Daniel T Infield ◽  
Jason D Galpin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Cyanine Dye ◽  
Nonsense Suppression ◽  
Xenopus Laevis Oocyte ◽  
Cellular Environment ◽  
Oocyte Expression System ◽  
Improved Technique

A general method is described for the site-specific genetic encoding of cyanine dyes as non-canonical amino acids (Cy-ncAAs) into proteins. The approach relies on an improved technique for nonsense suppression with in vitro misacylated orthogonal tRNA. The data show that Cy-ncAAs (based on Cy3 and Cy5) are tolerated by the eukaryotic ribosome in cell-free and whole-cell environments and can be incorporated into soluble and membrane proteins. In the context of the Xenopus laevis oocyte expression system, this technique yields ion channels with encoded Cy-ncAAs that are trafficked to the plasma membrane where they display robust function and distinct fluorescent signals as detected by TIRF microscopy. This is the first demonstration of an encoded cyanine dye as a ncAA in a eukaryotic expression system and opens the door for the analysis of proteins with single-molecule resolution in a cellular environment.

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