CLIC1 is a member of the CLIC family of proteins, which has been shown to demonstrate chloride channel activity when reconstituted in phospholipid vesicles. CLIC1 exists in cells as an integral membrane protein and as a soluble cytoplasmic protein, implying that CLIC1 might cycle between membrane-inserted and soluble forms. CLIC1 was purified and detergent was removed, yielding an aqueous solution of essentially pure protein. Pure CLIC1 was mixed with vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1 confers anion channel activity to preformed membranes that is indistinguishable from the previously reported activity resulting from reconstitution of CLIC1 into membranes by detergent dialysis. The activity is dependent on the amount of CLIC1 added, appears rapidly on mixing of protein and lipid, is inhibited by indanyloxyacetic acid-94, N-ethylmaleimide, and glutathione, is inactivated by heat, and shows sensitivity to pH and to membrane lipid composition. We conclude that CLIC1 in the absence of detergent spontaneously inserts into preformed membranes, where it can function as an anion-selective channel.
p64 is a chloride channel of intracellular membranes which is present in regulated secretory vesicles. Mechanisms by which the p64 channel could be regulated are largely unknown. p59fyn is a non-receptor tyrosine kinase of the Src family that has been implicated in a variety of intracellular signaling events. The N-terminal portion of p64 has several potential binding sites for Src family SH2 domains. In this paper, we demonstrate that p64 becomes tyrosine phosphorylated when co-expressed with p59 fyn in HeLa cells. We show that co-expression of p64 with p59 fyn renders p64 a ligand for the SH2 domain of p59 fyn and this SH2 binding is eliminated by treating p64 with alkaline phosphatase. Using sitedirected mutagenesis, we find that tyrosine 33 in the p64 sequence is necessary for SH2 binding. We also characterized p64-p59 fyn interactions using native material from bovine kidney. We found that a small fraction of native kidney p64 can bind Fyn SH2 in vitro. Immunoprecipitation of p64 from solubilized kidney membranes yields a kinase activity with the same mobility by SDSpolyacrylamide gel electrophoresis as authentic bovine p59fyn . Finally, we demonstrate that co-expression of p64 and p59 fyn in HeLa cells results in enhanced p64-associated chloride channel activity.
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