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

• Published in: Journal of cell science Vol. 122; no. 6; pp. 813 - 821
• Main Authors: Crane, Jonathan M, Verkman, Alan S
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists Limited 15.03.2009; Company of Biologists
• Subjects: Amino Acid Sequence; Animals; Aquaporin 1 - chemistry; Aquaporin 4 - chemistry; Aquaporin 4 - metabolism; Cell Membrane - metabolism; Cell Survival; Cercopithecus aethiops; COS Cells; Diffusion; Humans; Imaging, Three-Dimensional - methods; Microscopy, Fluorescence - methods; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Rats; Sequence Deletion
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.042341

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.