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J. Biol. Chem. 281 (40): 29448-29454

© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

The Putative Chloride Channel hCLCA2 Has a Single C-terminal Transmembrane Segment*

Randolph C. Elble{ddagger}1, Vijay Walia{ddagger}, Hung-chi Cheng§, Che J. Connon, Lars Mundhenk||, Achim D. Gruber||, , and Bendicht U. Pauli**

{ddagger}Department of Pharmacology and Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois 62794-9629, §Department of Biochemistry and Molecular Biology, Medical College, National Cheng Kung University, Tainan 70101, Taiwan, School of Optometry and Vision Sciences, Cardiff University, Cardiff CF23 9BD, United Kingdom, ||Department of Veterinary Pathology, Free University of Berlin, Robert-von-Ostertag-Strasse 15, D-14163 Berlin, Germany, and **Department of Molecular Medicine, Cornell University, Ithaca, New York 14853

Abstract: Calcium-activated chloride channel (CLCA) proteins were first described as a family of plasma membrane Cl channels that could be activated by calcium. Genetic and electrophysiological studies have supported this view. The human CLCA2 protein is expressed as a 943-amino-acid precursor whose N-terminal signal sequence is removed followed by internal cleavage near amino acid position 680. Earlier investigations of transmembrane geometry suggested five membrane passes. However, analysis by the more recently derived simple modular architecture research tool algorithm predicts that a C-terminal 22-amino-acid hydrophobic segment comprises the only transmembrane pass. To resolve this question, we raised an antibody against hCLCA2 and investigated the synthesis, localization, maturation, and topology of the protein. Cell surface biotinylation and endoglycosidase H analysis revealed a 128-kDa precursor confined to the endoplasmic reticulum and a maturely glycosylated 141-kDa precursor at the cell surface by 48 h post-transfection. By 72 h, 109-kDa N-terminal and 35-kDa C-terminal cleavage products were detected at the cell surface but not in the endoplasmic reticulum. Surprisingly, however, the 109-kDa product was spontaneously shed into the medium or removed by acid washes, whereas the precursor and 35-kDa product were retained by the membrane. Two other CLCA family members, bCLCA2 and hCLCA1, also demonstrated preferential release of the N-terminal product. Transfer of the hCLCA2 C-terminal hydrophobic segment to a secreted form of green fluorescent protein was sufficient to target that protein to the plasma membrane. Together, these data indicate that hCLCA2 is mostly extracellular with only a single transmembrane segment followed by a short cytoplasmic tail and is itself unlikely to form a channel.


Received for publication June 20, 2006. Revision received July 20, 2006.

* This work was supported by Philip Morris USA Inc., Philip Morris International, and by United States Army Breast Cancer Research Fund Grant DAMD17-00-1-0219 (to R. C. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed. Tel.: 217-545-7381; E-mail: relble2{at}siumed.edu.


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