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First published online 24 February 2009
doi: 10.1242/jcs.042341

Journal of Cell Science 122, 813-821 (2009)
Published by The Company of Biologists 2009
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Research Article

Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging

Jonathan M. Crane and Alan S. Verkman*

Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA

* Author for correspondence (e-mail: Alan.Verkman{at}ucsf.edu)

Accepted 17 November 2008

We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length `long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the `short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23.

Key words: AQP4, OAP, Water channel, Water permeability, Single particle tracking


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