Aquaporin-1 (AQP1) is the prototype integral membrane protein water channel. Although the three-dimensional structure and water transport function of the molecule have been described, the physical interactions between AQP1 and other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracellular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and fluorescence imaged microdeformation (FIMD) techniques were; performed on intact human red cells. By FPR, the fractional mobility of fluorescently labeled AQP1 (F-αAQP1) in the undeformed red cell membrane is 66 ± 10% and the average lateral diffusion coefficient is (3.1 ± 0.5) x 10-11 cm2/s. F-αAQP1 fractional mobility is not significantly affected by antibody-induced immobilization of the major integral proteins band 3 or glycophorin A, indicating that AQP1 does not exist as a complex with these proteins. FIMD uses pipette aspiration of individual red cells to create a constant but reversible skeletal density gradient. F-∅AQP1 distribution, like that of lipid-anchored proteins, is not at equilibrium after microdeformation. Over time, ~50% of the aspirated F-αAQP1 molecules migrate toward the membrane portion that had been maximally dilated, the aspirated cap. Based on the kinetics of migration, the F-αAQP1 lateral diffusion coefficient in the membrane projection is estimated to be 6 x 10- 10 cm2/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectrin- based membrane skeleton and/or by skeleton-linked membrane components, and that release of these constraints by dilatation of the skeleton allows AQP1 to diffuse much more rapidly in the plane of the membrane.
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