Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)–dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference–mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.

Vasopressin-regulated water reabsorption through the water channel aquaporin-2 (AQP2) in renal collecting ducts maintains body water homeostasis. Vasopressin activates PKA, which phosphorylates AQP2, and this phosphorylation event is required to increase the water permeability and water reabsorption of the collecting duct cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. However, whether this phosphorylation regulates the water permeability of this channel still remains unclear. To clarify the role of AQP2 phosphorylation in water permeability, we expressed recombinant human AQP2 in Escherichia coli, purified it, and reconstituted it into proteoliposomes. AQP2 proteins not reconstituted into liposomes were removed by fractionating on density step gradients. AQP2-reconstituted liposomes were then extruded through polycarbonate filters to obtain unilamellar vesicles. PKA phosphorylation significantly increased the osmotic water permeability of AQP2-reconstituted liposomes. We then examined the roles of AQP2 phosphorylation at Ser-256 and Ser-261 in the regulation of water permeability using phosphorylation mutants reconstituted into proteoliposomes. The water permeability of the non-phosphorylationmimicking mutant S256A-AQP2 and non-phosphorylated WT-AQP2 was similar, and that of the phosphorylation-mimicking mutant S256D-AQP2 and phosphorylated WT-AQP2 was similar. The water permeability of S261A-AQP2 and S261D-AQP2 was similar to that of non-phosphorylated WT-AQP2. This study shows that PKA phosphorylation of AQP2 at Ser-256 enhances its water permeability.Body water homeostasis is essential for the survival of mammals and is regulated by the renal collecting duct. Key components in the regulation of collecting duct water permeability are the vasopressin receptor and the water channel aquaporin-2 (AQP2) 2 (1-6). Mutations in the vasopressin V 2 receptor and AQP2 cause congenital nephrogenic diabetes insipidus, a disease characterized by a massive loss of water through the kidney (7,8). The binding of the antidiuretic hormone vasopressin to vasopressin V 2 receptors on renal principal cells stimulates cAMP synthesis via activation of adenylate cyclase. The subsequent activation of PKA leads to phosphorylation of AQP2 at Ser-256, and this phosphorylation event is required to increase the water permeability and water reabsorption of renal principal cells. It has been established that the phosphorylation of AQP2 induces its apical membrane insertion, rendering the cell water-permeable. We showed recently that AQP2 binds to a multiprotein "motor" complex and that phosphorylation-dependent reciprocal interaction with G-actin and tropomyosin 5b directs AQP2 to the apical membrane (9 -13). However, whether this phosphorylation regulates the water permeability of individual AQP2 proteins still remains unclear. Kuwahara et al. (14) showed that cAMP stimulation increased the water permeability of AQP2 expressed in Xenopus oocytes, alt...

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