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A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters
Published on: April 20, 2015
The putative chloride channel hCLCA2 has a single C-terminal transmembrane segment
Randolph C Elble1, Vijay Walia, Hung-Chi Cheng
1Department of Pharmacology and Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629, USA. relble2@siumed.edu
The Journal of Biological Chemistry
|July 29, 2006
Summary
Human CLCA2 protein has a single transmembrane segment, with its N-terminal portion shed from the cell surface. This suggests CLCA2 is extracellular and unlikely to form a channel.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Calcium-activated chloride channel (CLCA) proteins were initially characterized as plasma membrane channels activated by calcium.
- Previous studies suggested multiple transmembrane passes for human CLCA2 (hCLCA2).
Purpose of the Study:
- To resolve the transmembrane topology of hCLCA2.
- To investigate the synthesis, localization, maturation, and topology of hCLCA2.
Main Methods:
- Antibody generation against hCLCA2.
- Cell surface biotinylation and endoglycosidase H analysis.
- Analysis of protein cleavage products and their localization.
Main Results:
- hCLCA2 matures into a 141-kDa form at the cell surface.
- Cleavage yields 109-kDa N-terminal and 35-kDa C-terminal products at the cell surface.
- The N-terminal product is shed, while the C-terminal product remains membrane-associated.
- The C-terminal hydrophobic segment targets a reporter protein to the plasma membrane.
Conclusions:
- hCLCA2 possesses a single transmembrane segment and is predominantly extracellular.
- The N-terminal domain is released, and hCLCA2 is unlikely to function as a channel itself.
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