Phosphorylation of aquaporin-2 does not alter the membrane water permeability of rat papillary water channel-containing vesicles.
Antidiuretic hormone modulates the water permeability (Pf) of epithelial cells in the rat kidney by vesicle-mediated insertion and removal of the aquaporin-2 (AQP-2) water channel. AQP-2 possesses a single consensus cAMP-dependent protein kinase A (PKA) phosphorylation site (Ser-256) hypothesized to regulate channel Pf(Kuwahara, M., Fushimi, K., Terada, Y., Bai, L., Sasaki, S., and Marumo, F. (1995) J. Biol. Chem. 270, 10384-10387). To test whether PKA phosphorylation of AQP-2 alters channel Pf, we compared the Pf values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [gamma-32P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 32P label from AQP-2. However, the Pf (0.16 +/- 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 +/- 0. 3 mol of PO4/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 +/- 8% of the phosphate has been removed (Pf 0.14 +/- 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's Pf. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle compartments.
Lande
M B
MB
Division of Nephrology, Children's Hospital, Boston, Massachusetts 02115, USA.
Jo
I
I
Zeidel
M L
ML
Somers
M
M
Harris
H W
HW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001834:Body Water / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism*; D005260:Female; D007473:Ion Channels / Q000302:isolation & purification / Q000378:metabolism*; D007679:Kidney Medulla / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D013997:Time Factors
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D007473:Ion Channels; D010750:Phosphoproteins; D012694:Serine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases