FUNCTIONAL EXPRESSION OF P64, AN INTRACELLULAR CHLORIDE CHANNEL PROTEIN

p64 is a protein identified as a chloride channel by biochemical purif ication from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cel l membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found i n cells expressing p64 and not in control cells. The p64-associated ac tivity shows strong anion over cation selectivity. Single channels sho w prominent outward rectification with single channel conductance at p ositive potentials of 42 pS. The chloride channel activity is activate d by treatment of the membranes with alkaline phosphatase and inhibite d by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeabilit y was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increa se in chloride permeability, consistent with expression of a novel chl oride channel activity.