2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

Conference: 2000 Global Researcher Conference
Title: The stoichiometry of phosphorylated and non-phosphorylated monomers in an aquaporin-2 tetramer determines its subcellular localization
Authors: Kamsteeg, Erik-Jan; Heijnen, I.; van Os, Carel; Deen, Peter M.T.
Institution: University of Nijmegen
Kamsteeg In renal collecting duct principal cells, phosphorylation of the aquaporin-2 (AQP2) water channel by protein kinase A is essential for its redistribution from vesicles to the apical membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in vesicles of principal cells. Since AQP2 is expressed as homotetramers, the number of p-AQP2 monomers in a tetramer might be critical for this redistribution. Expressed in oocytes, AQP2-S256A and AQP2-S256D mimicked non-p-AQP2 and p-AQP2, respectively, since routing and function of AQP2-S256D and wt-AQP2 were identical whereas AQP2-S256A was retained intracellularly. Also, AQP2-S256A and AQP2-S256D formed heterotetramers. Subsequent co-injection of different ratios of AQP2-S256A and AQP2-S256D cRNAs clearly revealed that at least three AQP2-S256D monomers in an AQP2 tetramer were essential for plasma membrane localization of AQP2. Therefore, our results strongly indicate that in principal cells, three monomers per tetramer have to be phosphorylated for its apical membrane localization. Also, since other multi-subunit channels are regulated by phosphorylation, it is likely that the stoichiometry of phosphorylated and non-phosphorylated subunits also fine-tune the activity or subcellular localization of these complexes.

In order for AQP2 to leave their intracellular transport sacs and get to the apical cell membrane, it must first undergo phosphorylation, a process that connects it to a phosphate group. Nonetheless, phosphorylated AQP2 (p-AQP2) has been detected inside vesicles (the transport sacs) as well as the apical membrane. Since AQP2 is made up of a union of four like parts, the researchers speculated that the number of parts comprising the AQP2 that get phosphorylated might be the determining factor in its movement from the vesicle to the apical membrane.

The researchers injected two different AQP2 mutants into laboratory cell cultures. One mutant acted like non-phosphorylated AQP2 (non-p-AQP2); the other acted as p-AQP2. The non-p-AQP2 mutant is retained in the cell, never traveling to the apical membrane; the mutant p-AQP2 does. Also, neither of the mutants was made up of four like parts. Subsequent testing suggests that at least three out of the four parts that comprise an AQP2 must undergo phosphorylation to be able to move from the vesicles to the apical membrane. The researchers further suggest that the chemical changes of phosphorylated and non-phosphorylated sub-units of other channel proteins effect the activity of sub-cellular localization of the proteins they comprise.