Phosphorylation of Aquaporin-2 Does Not Alter the Membrane Water Permeability of Rat Papillary Water Channel-containing Vesicles (*)
- Marc B. Lande(1)(§),
- Inho Jo(1),
- Mark L. Zeidel(2)(¶),
- Michael Somers(1) and
- H. William Jr. Harris(1)(**)
- From the (1)Division of Nephrology, Children's Hospital, Boston, Massachusetts 02115 and the
- (2)Department of Medicine, Laboratory of Epithelial Cell Biology, University of Pittsburgh, Presbyterian University Hospital, Pittsburgh, Pennsylvania 15213
- **Established Investigator of the American Heart Association. To whom correspondence should be addressed: Div. of Nephrology, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-735-6526; Fax: 617-730-0435.
Abstract
Antidiuretic hormone modulates the water permeability (P
) 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 P
(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 P
, we compared the P
values of purified AQP-2 endosomes after incubation with either PKA or alkaline phosphatase. Studies using [
-
P]ATP reveal that AQP-2 endosomes contain endogenous PKA and phosphatase activities that add and remove 
P label from AQP-2. However, the P
(0.16 ± 0.06 cm/s) of endosomes containing phosphorylated AQP-2 (0.7 ± 0.3 mol of PO
/mol of protein) is not significantly different from the same AQP-2 endosomes where 95 ± 8% of the phosphate has been removed
(P
0.14 ± 0.06 cm/s). These data do not support a role for PKA phosphorylation in alteration of AQP-2's P
. Instead, AQP-2 phosphorylation by PKA may modulate AQP-2's distribution between plasma membrane and intracellular vesicle
compartments.
Footnotes
-
↵¶ Recipient of career development and merit review awards from the Medical Research Service of the Department of Veterans Affairs.
-
↵* This work was supported in part by National Institutes of Health Grants DK38874, 43955, and HL15157. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- ADH
-
antidiuretic hormone
- AQP
-
aquaporin
- IMCD
-
inner medullary collecting duct
- PKA
-
cAMP-dependent protein kinase A
- F-dextran
-
fluorescein isothiocyanate-conjugated dextran
- J
-
membrane water flux
- P
-
membrane osmotic water permeability
- IP
-
20-mer peptide that is a specific inhibitor of cAMP-dependent protein kinase A activity
- CAPS
-
3-(cyclohexylamino)propanesulfonic acid.
-
- Received December 9, 1995.
- Revision received December 25, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
