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Sodium channels in membrane vesicles from cultured toad bladder cells
C Asher1, A Moran, B C Rossier
1Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
The American Journal of Physiology
|April 1, 1988
Summary
This study shows that TBM-18(c123) cells can model epithelial sodium channels (ENaC). Aldosterone treatment increased amiloride-sensitive sodium flux, indicating hormonal regulation of these channels.
Area of Science:
- Cell biology
- Molecular physiology
- Ion channel research
Background:
- The epithelial sodium channel (ENaC) plays a critical role in sodium reabsorption in various tissues.
- Understanding ENaC regulation is crucial for managing conditions related to fluid and electrolyte balance.
- Cultured cell models offer a valuable tool for studying ion channel function and regulation.
Purpose of the Study:
- To characterize the amiloride-sensitive sodium (22Na+) flux in TBM-18(c123) cells.
- To investigate the presence of calcium-dependent regulation of channel conductance in these cells.
- To determine the effect of aldosterone on the epithelial sodium channel in a cellular model.
Main Methods:
- Preparation of membrane vesicles from TBM-18(c123) cells cultured on porous supports.
- Measurement of electrical potential-driven 22Na+ fluxes using tracer uptake assays.
- Assessment of amiloride inhibition to quantify amiloride-sensitive flux and determine inhibition constants.
- Incubation of cells with aldosterone to evaluate hormonal effects on channel activity.
Main Results:
- A significant portion (50-70%) of 22Na+ uptake in vesicles from cells on porous supports was inhibited by amiloride (Ki < 0.1 microM), indicating ENaC mediation.
- Amiloride-sensitive fluxes were substantially lower in vesicles from cells not grown on porous supports.
- Calcium-dependent processes down-regulating channel conductance were observed, similar to native epithelia.
- Aldosterone preincubation (5 x 10(-7) M for 16-20 h) significantly increased amiloride-sensitive conductance compared to control cells.
Conclusions:
- TBM-18(c123) cell-derived membranes serve as a valid model for characterizing the epithelial sodium channel.
- The study confirms the presence of key regulatory mechanisms, including calcium-dependent processes and aldosterone stimulation, in this cellular model.
- This model can be utilized to further investigate ENaC function and its hormonal regulation in a controlled in vitro setting.