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Title: Single-channel properties and regulation of chloride intracellular channel proteins
Author: Singh, H.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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In this study, soluble recombinant, human CLIC1, rat brain CLIC4 (p64H1) and human CLIC5A, a splice variant of p64 (CLIC5B), were expressed as cleavable His-tagged proteins and incorporated into voltage-clamped planar lipid bilayers. They inserted spontaneously with or without an intact His-tag to form redox-regulated ion channels. The putative pore forming region was identified based on previous work and CLIC channels appear to contain at least 4 subunits each with a single transmembrane domain (TMD). In this simple model, the luminal side of each subunit contains a single cysteine residue located just before the putative pore entrance. Consistent with these ideas, truncated proteins comprising the first 58 residues or CLIC1, or the first 61 residues of CLIC4 (sufficient in each case to contain the putative TMD), autoinserted into bilayers to form redox-sensitive ion channels and could be blocked by cysteine reactive compounds applied from the luminal side. The truncated proteins showed reduced conductances, and were non-selective between anions and cations. CLIC5A and CLIC1 channel function may be regulated by the cortical actin cytoskeleton, providing a new mechanism to regulate localised ion flux in cells. Unusually, the interaction may be direct, without any intermediate or adapter protein. CLICs also interact with dynamin I and cytoskeletal proteins in vitro. The localisation of CLICs in nerve terminals and their interaction with dynamin suggests a possible role in SV recycling. These preliminary structure-function studies have provided an overview of the molecular mechanism of membrane CLICs. Discovery of regulatory mechanisms for CLICs may shed more light on their functional roles in cells during processes such as the cell cycle and apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available