Long-range nonanomalous diffusion of quantum dot-labeled aquaporin-1 water channels in the cell plasma membrane.
Aquaporin-1 (AQP1) is an integral membrane protein that facilitates osmotic water transport across cell plasma membranes in epithelia and endothelia. AQP1 has no known specific interactions with cytoplasmic or membrane proteins, but its recovery in a detergent-insoluble membrane fraction has suggested possible raft association. We tracked the membrane diffusion of AQP1 molecules labeled with quantum dots at an engineered external epitope at frame rates up to 91 Hz and over times up to 6 min. In transfected COS-7 cells, >75% of AQP1 molecules diffused freely over approximately 7 mum in 5 min, with diffusion coefficient, D(1-3) approximately 9 x 10(-10) cm(2)/s. In MDCK cells, approximately 60% of AQP1 diffused freely, with D(1-3) approximately 3 x 10(-10) cm(2)/s. The determinants of AQP1 diffusion were investigated by measurements of AQP1 diffusion following skeletal disruption (latrunculin B), lipid/raft perturbations (cyclodextrin and sphingomyelinase), and bleb formation. We found that cytoskeletal disruption had no effect on AQP1 diffusion in the plasma membrane, but that diffusion was increased greater than fourfold in protein de-enriched blebs. Cholesterol depletion in MDCK cells greatly restricted AQP1 diffusion, consistent with the formation of a network of solid-like barriers in the membrane. These results establish the nature and determinants of AQP1 diffusion in cell plasma membranes and demonstrate long-range nonanomalous diffusion of AQP1, challenging the prevailing view of universally anomalous diffusion of integral membrane proteins, and providing evidence against the accumulation of AQP1 in lipid rafts.
Crane
Jonathan M
JM
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
Verkman
A S
AS
D000818:Animals; D051398:Aquaporin 1 / Q000378:metabolism*; D020346:Aquaporins / Q000378:metabolism*; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004058:Diffusion; D004285:Dogs; D045663:Quantum Dots
C496020:AQP1 protein, human; D020346:Aquaporins; D051398:Aquaporin 1