title: Evaluation of the membrane-spanning domain of ClC-2.
abstract: The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure-function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.
authors.affiliation: Programme of Structural Biology and Biochemistry, Research Institute of the Hospital for Sick Children, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
authors.forename: Mohabir
authors.initials: M
authors.lastname: Ramjeesingh
authors.forename: Canhui
authors.initials: C
authors.lastname: Li
authors.forename: Yi-Min
authors.initials: YM
authors.lastname: She
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C504424:Alexa Fluor 488 C5-maleimide; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D008081:Liposomes; D008301:Maleimides; D003545:Cysteine