4.5 Article

Reversible, Temperature-Dependent Supramolecular Assembly of Aquaporin-4 Orthogonal Arrays in Live Cell Membranes

期刊

BIOPHYSICAL JOURNAL
卷 97, 期 11, 页码 3010-3018

出版社

CELL PRESS
DOI: 10.1016/j.bpj.2009.09.017

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资金

  1. National Institutes of Health [EB00415, HL73856, DK35124, EY13574, DK86125, DK72517]
  2. Guthy-Jackson Charitable Foundation
  3. Research Development Program and Drug Discovery
  4. Cystic Fibrosis Foundation
  5. National Institutes of Health, National Research Service award [GM808512]

向作者/读者索取更多资源

The shorter M23' isoform of the glial cell water channel aquaporin-4 (AQP4) assembles into orthogonal arrays of particles (OAPs) in cell plasma membranes, whereas the full-length M1 isoform does not. N-terminal residues are responsible for OAP formation by AQP4-M23 and for blocking of OAP formation in AQP4-M1. In investigating differences in CAP formation by certain N-terminus mutants of AQP4, as measured by freeze-fracture electron microscopy versus live-cell imaging, we discovered reversible, temperature-dependent OAP assembly of certain weakly associating AQP4 mutants. Single-particle tracking of quantum-dot-labeled AQP4 in live cells and total internal reflection fluorescence microscopy showed >80% of M23 in OAPs at 10-50 degrees C compared to <10% of M1. However, OAP formation by N-terminus cysteine-substitution mutants of M1, which probe palmitoylation-regulated OAP assembly, was strongly temperature-dependent, increasing from <10% at 37 degrees C to >70% at 10 degrees C for the double mutant M1-C13A/C17A. OAP assembly by this mutant, but not by native M23, could also be modulated by reducing its membrane density. Exposure of native M1 and single cysteine mutants to 2-bromopalmitate confirmed the presence of regulated CAP assembly by S-palmitoylation. Kinetic studies showed rapid and reversible CAP formation during cooling and OAP disassembly during heating. Our results provide what to our knowledge is the first information on the energetics of AQP4 OAP assembly in plasma membranes.

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