Nucleotides bind to the C-terminus of ClC-5.

Abstract

Mutations in ClC-5 (chloride channel 5), a member of the ClC family of chloride ion channels and antiporters, have been linked to Dent's disease, a renal disease associated with proteinuria. Several of the disease-causing mutations are premature stop mutations which lead to truncation of the C-terminus, pointing to the functional significance of this region. The C-terminus of ClC-5, like that of other eukaryotic ClC proteins, is cytoplasmic and contains a pair of CBS (cystathionine beta-synthase) domains connected by an intervening sequence. The presence of CBS domains implies a regulatory role for nucleotide interaction based on studies of other unrelated proteins bearing these domains [Ignoul and Eggermont (2005) Am. J. Physiol. Cell Physiol. 289, C1369-C1378; Scott, Hawley, Green, Anis, Stewart, Scullion, Norman and Hardie (2004) J. Clin. Invest. 113, 274-284]. However, to date, there has been no direct biochemical or biophysical evidence to support nucleotide interaction with ClC-5. In the present study, we have expressed and purified milligram quantities of the isolated C-terminus of ClC-5 (CIC-5 Ct). CD studies show that the protein is compact, with predominantly alpha-helical structure. We determined, using radiolabelled ATP, that this nucleotide binds the folded protein with low affinity, in the millimolar range, and that this interaction can be competed with 1 muM AMP. CD studies show that binding of these nucleotides causes no significant change in secondary structure, consistent with a model wherein these nucleotides bind to a preformed site. However, both nucleotides induce an increase in thermal stability of ClC-5 Ct, supporting the suggestion that both nucleotides interact with and modify the biophysical properties of this protein.

• Authors: Leigh Wellhauser, Hsin-Hen Kuo, Fiona L L Stratford, Mohabir Ramjeesingh, Ling-Jun Huan, Winnie Luong, Canhui Li, Charles M Deber, Christine E Bear
• Journal: The Biochemical journal (Biochem J) Vol. 398 Issue 2 Pg. 289-94 (Sep 01 2006) ISSN: 1470-8728 [Electronic] England
• PMID: 16686597 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Chloride Channels
• Recombinant Proteins
• Adenosine Monophosphate
• Adenosine Triphosphate
• Adenosine Triphosphatases

Topics

• Adenosine Monophosphate (chemistry, metabolism)
• Adenosine Triphosphatases (metabolism)
• Adenosine Triphosphate (chemistry, metabolism)
• Chloride Channels (chemistry, genetics, isolation & purification, metabolism)
• Circular Dichroism
• Gene Expression
• Humans
• Hydrolysis
• Protein Denaturation
• Protein Structure, Secondary
• Recombinant Proteins (chemistry, genetics, isolation & purification, metabolism)
• Temperature