Functional Reconstitution of the Human Epithelial Na+ Channel in a Mammalian Expression System

Ion Channels ◽  
2006 ◽  
pp. 3-13
Author(s):  
Alexander Staruschenko ◽  
Rachell E. Booth ◽  
Oleh Pochynyuk ◽  
James D. Stockand ◽  
Qiusheng Tong
Keyword(s):  
Expression System ◽  
Na Channel ◽  
Mammalian Expression ◽  
Mammalian Expression System ◽  
Epithelial Na Channel

Optimization of a mammalian expression system for the measurement of sodium channel gating currents

1996 ◽  
Vol 271 (3) ◽  
pp. C1001-C1006 ◽  
Author(s):  
M. F. Sheets ◽  
J. W. Kyle ◽  
S. Krueger ◽  
D. A. Hanck
Keyword(s):  
Membrane Surface ◽  
Expression System ◽  
Channel Gating ◽  
Na Channel ◽  
Na Channels ◽  
Gating Currents ◽  
Small Magnitude ◽  
Electrical Signals ◽  
Mammalian Expression ◽  
Mammalian Expression System

We describe a new mammalian expression system that optimizes conditions for the measurement of Na channel gating currents (Ig). The small magnitude of Ig limits their study to preparations with high numbers of Na channels to improve signal-to-noise ratios. To increase Na channel Ig signals, single tsA201 cells (approximately 20 microns in diameter) were fused into large, multinucleated cells by treatment with polyethylene glycol. After being placed in cell culture for 48-72 h, fused tsA201 cells develop a spherical geometry with diameters up to 200 microns. Because of the large plasma membrane surface area, fused tsA201 cells are able to express high levels of Na channels after transient transfection with Na channel cDNAs using Lipofectamine. Typically, 5 days after transfection, fused tsA201 cells that are 60-100 microns in diameter are selected for voltage clamp with a large suction pipette (a pore size of 20-30 microns) that allows for both a low series resistance and internal perfusion. Approximately two-thirds of transfected fused tsA201 cells express Na current, with nearly one-third of transfected cells expressing sufficient numbers of Na channels to allow for the ready measurement of Ig. In addition to fused tsA201 cells being a preparation well suited for the study of Ig, they should also be useful for measurement of electrical signals from other voltage-gated channels and transporters that generate small electrical signals.

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 Regulation of the Epithelial Na+ Channel by Membrane Tension

1998 ◽  
Vol 112 (2) ◽  
pp. 97-111 ◽  
Author(s):  
Mouhamed S. Awayda ◽  
Muthangi Subramanyam
Keyword(s):  
Cytochalasin B ◽  
Single Channel ◽  
Direct Injection ◽  
Expression System ◽  
Cell Swelling ◽  
Na Channel ◽  
Bathing Solution ◽  
Membrane Tension ◽  
Whole Cell ◽  
Epithelial Na Channel

The sensitivity of αβγ rat epithelial Na+ channel (rENaC) to osmotically or mechanically induced changes of membrane tension was investigated in the Xenopus oocyte expression system, using both dual electrode voltage clamp and cell-attached patch clamp methodologies. ENaC whole-cell currents were insensitive to mechanical cell swelling caused by direct injection of 90 or 180 nl of 100-mM KCl. Similarly, ENaC whole-cell currents were insensitive to osmotic cell swelling caused by a 33% decrease of bathing solution osmolarity. The lack of an effect of cell swelling on ENaC was independent of the status of the actin cytoskeleton, as ENaC remained insensitive to osmotic and mechanical cell swelling in oocytes pretreated with cytochalasin B for 2–5 h. This apparent insensitivity of ENaC to increased cell volume and changes of membrane tension was also observed at the single channel level in membrane patches subjected to negative or positive pressures of 5 or 10 in. of water. However, and contrary to the lack of an effect of cell swelling, ENaC currents were inhibited by cell shrinking. A 45-min incubation in a 260-mosmol solution (a 25% increase of solution osmolarity) caused a decrease of ENaC currents (at −100 mV) from −3.42 ± 0.34 to −2.02 ± 0.23 μA (n = 6). This decrease of current with cell shrinking was completely blocked by pretreatment of oocytes with cytochalasin B, indicating that these changes of current are not likely related to a direct effect of cell shrinking. We conclude that αβγ rENaC is not directly mechanosensitive when expressed in a system that can produce a channel with identical properties to those found in native epithelia.

scholarly journals Glycosylation in a Mammalian Expression System Is Critical for the Production of Functionally Active Leukocyte Immunoglobulin-like Receptor A3 Protein

2013 ◽  
Vol 288 (46) ◽  
pp. 32873-32885 ◽  
Author(s):  
Terry H. Y. Lee ◽  
Ainslie Mitchell ◽  
Sydney Liu Lau ◽  
Hongyan An ◽  
Poornima Rajeaskariah ◽  
...  
Keyword(s):  
Expression System ◽  
Mammalian Expression ◽  
Mammalian Expression System

Functional Reconstitution of the Human Epithelial Na+ Channel in a Mammalian Expression System

Ion Channels ◽  
2006 ◽  
pp. 1-114
Author(s):  
Alexander Staruschenko ◽  
Rachell E. Booth ◽  
Oleh Pochynyuk ◽  
James D. Stockand ◽  
Qiusheng Tong
Keyword(s):  
Expression System ◽  
Mammalian Expression ◽  
Mammalian Expression System

A NOVEL MAMMALIAN EXPRESSION SYSTEM BASED ON THE GLOBIN LOCUS CONTROL REGION

1992 ◽  
pp. 68-70
Author(s):  
J McLean ◽  
M Hollis ◽  
M Needham ◽  
C Gooding ◽  
K Hudson ◽  
...  
Keyword(s):  
Control Region ◽  
Expression System ◽  
Locus Control Region ◽  
Mammalian Expression ◽  
Mammalian Expression System
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