LONG-RANGE NON-ANOMALOUS DIFFUSION OF QUANTUM DOT-LABELED AQUAPORIN-1 WATER CHANNELS IN THE CELL PLASMA MEMBRANE

¹ University of California San Francisco

^* To whom correspondence should be addressed. E-mail: verkman{at}itsa.ucsf.edu.

Submitted on June 14, 2007
Revised on July 31, 2007
Accepted on 7 September 2007

	Abstract

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 detergentinsoluble 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 ~7 µm in 5 min, with diffusion coefficient, D_1-3 ~ 9 x 10^-10 cm²/s. In MDCK cells, ~60 % of AQP1 diffused freely, with D_1-3 ~ 3 x 10^-10 cm²/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 >4-fold 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.

Key Words: AQP1, Aquaporin, anomalous diffusion, photobleaching, single particle tracking

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