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An epithelial high-affinity amiloride-binding site, different from the Na+ channel
O Goldstein1, C Asher, P Barbry
1Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
The Journal of Biological Chemistry
|April 15, 1993
Summary
Chicken intestine plasma membranes contain a high-affinity amiloride receptor distinct from the epithelial sodium channel. This receptor does not directly block sodium transport, suggesting a novel functional role.
Area of Science:
- Biochemistry
- Physiology
- Molecular Biology
Background:
- Amiloride and its analogues are crucial tools for studying ion transport.
- The epithelial sodium channel (ENaC) is a key target for amiloride binding and inhibition.
- Chicken lower intestine is an important model for studying epithelial transport.
Purpose of the Study:
- To characterize the specific binding sites of radioactive amiloride analogues ([3H]phenamil and [3H]benzamil) in chicken lower intestine plasma membranes.
- To compare the properties of amiloride binding sites with those involved in sodium (Na+) transport inhibition.
- To identify potential novel amiloride-binding proteins in epithelial tissues.
Main Methods:
- Radioligand binding assays using [3H]phenamil and [3H]benzamil.
- Competition binding studies with pyrazinecarboxamides, including 5-(N-ethyl-N-isopropyl)-amiloride.
- Comparison of binding kinetics with Na+ transport inhibition data.
Main Results:
- A single population of high-affinity amiloride binding sites was identified in chicken lower intestine plasma membranes.
- These binding sites exhibit affinities and specificities for pyrazinecarboxamides that resemble ENaC.
- However, 5-(N-ethyl-N-isopropyl)-amiloride displaced [3H]phenamil at 10-fold lower concentrations than required for Na+ channel block.
- Phenamil association and dissociation rates at this site were slower than Na+ channel inhibition/reactivation rates.
- Similar high-affinity amiloride binding sites were found in other epithelia.
Conclusions:
- Chicken lower intestine and other tight epithelia possess a major high-affinity amiloride receptor independent of the Na+ conducting channel.
- This novel receptor is not the Na+/H+ antiport or the Na+/Ca2+ exchanger.
- The receptor may represent non-conducting channels, a related channel subtype, or an unrelated protein, indicating a previously unrecognized role for amiloride binding in epithelial physiology.