Very High Single Channel Water Permeability of Aquaporin-4 in Baculovirus-Infected Insect Cells and Liposomes Reconstituted with Purified Aquaporin-4

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Abstract
The insect cell/baculovirus system was used to express the mercurial-insensitive water channel aquaporin-4 (AQP4) for purification and reconstitution. Immunoblot analysis of Sf9 cells infected with recombinant baculovirus showed greatest AQP4 expression at 72 h after infection at a multiplicity-of-infection of 5. Immunostaining and cell membrane fractionation indicated AQP4 plasma membrane expression. Quantitative immunoblot analysis showed approximately 60 microg of AQP4 per milligram of plasma membrane protein (approximately 2 mg of AQP4 protein per liter of Sf9 cell culture). Functional analysis by stopped-flow light scattering indicated that AQP4 functioned as a mercurial-insensitive water-selective transporter. Osmotic water permeability (Pf) in plasma membrane vesicles from AQP4-expressing Sf9 cells was very high (0.053 cm/s at 10 degrees C), weakly temperature dependent (activation energy, 4.5 kcal/mol), and not inhibited by HgCl2. The AQP4 single channel water permeability (p(f)), estimated from Pf and protein amount, was 19 x 10(-14) cm3/s. Purification of AQP4 to a single Coomassie blue-stained protein on SDS-PAGE (1300-fold over homogenate) was achieved by membrane fractionation, carbonate stripping of nonintegral proteins, solubilization in octyl-beta-glucoside, and anion exchange chromatography. AQP4 protein identity was confirmed by mass spectrometry. Reconstitution of purified AQP4 into proteoliposomes increased osmotic water permeability by >40-fold, giving a p(f) of 15 x 10(-14) cm3/s, remarkably greater than that of 4.9 x 10(-14) cm3/s measured in parallel for AQP1. These results establish the first purification of an aquaporin from a heterologous expression system. The high AQP4 p(f) suggests (a) significant functional differences among the aquaporins, (b) inadequacy of existing pore models to account for high water flow and water permselectivity, and (c) possible enhancement of water flow by AQP4 assembly in orthogonal arrays.

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