cAMP Regulated Membrane Diffusion of a Green Fluorescent Protein-Aquaporin 2 Chimera

Abstract

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.

Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus

“…Currently, we cannot explain why YFP-Zm7hvr and 110-YFP-Zm7hvr had such relatively small mobile fractions. In spite of their small mobile fractions, YFP-Zm7hvr and 110-YFP-Zm7hvr could be used in this experiment, since calculation of the diffusion coefficient of a mobile fraction is independent of the size of this fraction (see Methods), and the diffusion coefficients of YFP-Zm7hvr and 110-YFP-Zm7hvr in the plasma membrane outside the tubules (Table 1) were comparable to those of other plasma membrane-associated proteins (Adams et al, 1998;Haggie et al, 2003;Jans et al, 1990;Meissner & Haberlein, 2003;Tardin et al, 2003;Umenishi et al, 2000).…”

“…animal cells (>90 %; Adams et al, 1998;Haggie et al, 2003;Umenishi et al, 2000). The mobile fraction was comparable in both the plasma membrane surrounding the tubule and the plasma membrane outside the tubule, showing that the small mobile fractions were due to an intrinsic property of the plasma membrane interaction domain and not due to the presence of the tubule.…”

Increased Diffusional Mobility of CFTR at the Plasma Membrane after Deletion of Its C-terminal PDZ Binding Motif

“…The region of interest-integrated fluorescence values were normalized to unity for prebleach fluorescence and corrected for bleaching during acquisition of serial images (correction generally Ͻ2% per image acquisition). Absolute diffusion coefficients (D) were computed from the time course of corrected fluorescence using the analytical solution to the one-dimensional diffusion equation with appropriate initial and boundary conditions as described for the measurements of GFP-aquaporin diffusion in cell plasma membranes (38). Spot-photobleaching data was analyzed as described previously (36).…”

The prostaglandin E2 analogue sulprostone antagonizes vasopressin-induced antidiuresis through activation of Rho

“…Similarly, depolymerization of stress fibers induced by cytochalasin D allows AQP2 translocation without elevation of cAMP in IMCD and CD8 cells (Klussmann et al, 2001b;Tamma et al, 2001). In addition to its barrier function, F-actin may directly interact with AQP2 (Brown et al, 1998;Umenishi et al, 2000).…”