Membrane dynamics of the water transport protein aquaporin-1 in intact human red cells

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 an d other membrane components have not been characterized. Using fluorescein isothiocyanate-anti-Co3 (FITC-anti-Co3), a reagent specific for an extracel lular epitope on AQP1, the fluorescence photobleaching recovery (FPR) and f luorescence imaged microdeformation (FIMD) techniques were performed on int act human red cells. By FPR, the fractional mobility of fluorescently label ed AQP1 (F-alpha AQP1) in the undeformed red cell membrane is 66 +/- 10% an d the average lateral diffusion coefficient is (3.1 +/- 0.5) x 10(-11) cm(2 )/s. F-alpha AQP1 fractional mobility is not significantly affected by anti body-induced immobilization of the major integral proteins band 3 or glycop horin A, indicating that AQP1 does not exist as a complex with these protei ns. FIMD uses pipette aspiration of individual red cells to create a consta nt but reversible skeletal density gradient, F-alpha AQP1 distribution, lik e that of lipid-anchored proteins, is not at equilibrium after microdeforma tion. Over time, similar to 50% of the aspirated F-alpha AQP1 molecules mig rate toward the membrane portion that had been maximally dilated, the aspir ated cap. Based on the kinetics of migration, the F-alpha AQP1 lateral diff usion coefficient in the membrane projection is estimated to be 6 x 10(-10) cm(2)/s. These results suggest that AQP1 lateral mobility is regulated in the unperturbed membrane by passive steric hindrance imposed by the spectri n-based membrane skeleton and/or by skeleton-linked membrane components, an d that release of these constraints by dilatation of the skeleton allows AQ P1 to diffuse much more rapidly in the plane of the membrane.