Aquaporin-6 (AQP6), an intracellular water channel with anion conductance
Research field:Kidney physiology
Authors:Yasui M, Hazama A, Kwon T-H, Nielsen S, Guggino WB, Agre P
Address of presenting
author:		M. Yasui, Johns Hopkins University School of Medicine, Department of Biological Chemistry, 725 N. Wolfe St., Baltimore, MD21205, USA
E-mail:myasui@jhmi.edu
Phone:+1 410 955 3154
Fax:+1 410 955 3149
Text of abstract Introduction
Aquaporin water channel proteins are responsible for the high membrane water permeability of many plant and animal tissues. Mammalian aquaporins were previously believed to reside partially or entirely in plasma membranes and are constitutively active. Aquaporins were not known to be permeated by ions or charged solutes, and were not believed to undergo gating. AQP6 is very different from most other mammalian aquaporins, since it is known to have low baseline water permeability (Ma et al., Genomics 35: 543-550, 1996). Sequence analysis reveals AQP6 to be most closely related to AQP0, which has a low water permeability, and AQP2, which resides in intracellular vesicles in collecting duct principal cells. A strong clue to the function of AQP6 was provided when the protein was recently shown to be restricted to intracellular vesicles in renal epithelia including acid-secreting intercalated cells in collecting duct (Yasui et al., Proc. Natl., Acad. Sci. USA 96: 5808-5813, 1999).

Methods
Wild-type or mutants rat AQP6 were expressed in Xenopus laevis oocytes. Osmotic water pearmeability (Pf) was assessed by osmotic swelling of the oocytes. The current was recorded by two-electrode voltage clamp. Patches were excised from the oocytes in the outside-out configuration.

Results
When expressed in X. laevis oocytes, AQP6 exhibits low basal water permeability (Pf). Surprisingly, when treated with the known water channel inhibitor, Hg++, the Pf of AQP6 oocytes rapidly rises ~10 fold and is accompanied by ion conductance. AQP6 colocalizes with H+-ATPase in intracellular vesicles of acid secreting intercalated cells in renal collecting duct. At pH < 5.5, anion conductance is rapidly and reversibly activated in AQP6 oocytes. Site-directed mutation of lysine to glutamate at position 72 in the cytoplasmic mouth of the pore changes the cation/anion selectivity but leaves low pH activation intact, suggesting that ions pass through AQP6 itself rather than passing through associated channels. Gating behavior of AQP6 was further demonstrated by a single channel patch-clamp which revealed rapid flickering.

Conclusions
The data demonstrate unprecedented biophysical properties by an aquaporin, indicating that AQP6 does not function as a simple conduit for trans-epithelial water absorption or secretion but participates in diverse physiological processes, including vesicle acidification.

References
Ma, T., Yang, B., Kuo, W.L., Verkman, A.S. 1996. Genomics 35, 49-59
Yasui, M., Kwon, T-H., Knepper, M. A., Nielsen, S. & Agre, P. 1999. Proc. Natl. Acad. Sci. USA 96, 5808-5813.
Yasui, M., Hazama, A., Kwon, T-H., Nielsen, S., Guggino, W.B. & Agre, P. 1999. Nature 402, 184-187.

Keywords:gating, osmotic water permeability, anion permeability, vesicle acidification

Created 2000-05-02