ULTRASTRUCTURE, PHARMACOLOGICAL INHIBITION, AND TRANSPORT SELECTIVITY OF AQUAPORIN CHANNEL-FORMING INTEGRAL PROTEIN IN PROTEOLIPOSOMES

Reconstitution of highly purified aquaporin CHIP (channel-forming integral protein) into proteoliposomes was previously shown to confer high osmotic water permeability (P-f) to the membranes [Zeidel et al. (1992) Biochemistry 31, 7436-7440]. Here we report detailed ultrastructural, pharmacologic, and transport studies of human red cell CHIP in proteoliposomes. Freeze-fracture and transmission electron microscopy revealed a uniform distribution of CHIP which was incorporated into the membranes in both native and inverse orientations. Morphometric analysis of membranes reconstituted at three different concentrations of CHIP revealed that the intramembrane particles correspond to tetramers or possible higher order oligomers, and the P-f increased in direct proportion to the CHIP density. Proteolytic removal of the 4-kDa C-terminal cytoplasmic domain of CHIP did not alter the P-f or oligomerization in red cell membranes. CHIP exhibited a similar conductance for water when reconstituted into membranes of varied lipid compositions. The sensitivities of CHIP-mediated P-f to specific sulfhydryl reagents were identical to known sensitivities of red cell P-f, including a delayed response to p-(chloromercuri) benzenesulfonate. CHIP did not increase the permeability of the proteoliposome membranes to H+/OH- or NH3. These studies demonstrate that CHIP proteoliposomes exhibit all known characteristics of water channels in native red cells and therefore provide a defined system for biophysical analysis of transmembrane water movements.