The Mercurial Insensitive Water Channel (AQP-4) Forms Orthogonal Arrays in Stably Transfected Chinese Hamster Ovary Cells (*)
- From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521 and
- Department of Pathology and Renal Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114
- § To whom correspondence should be addressed: 1246 Health Sciences, East Tower, Cardiovascular Research Inst., University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; :verkman{at}itsa.ucsf.edu.
Abstract
The mercurial insensitive water channel (MIWC, AQP-4) is a water-selective transporter expressed at the basolateral plasma
membrane of principal cells in kidney collecting duct, airway epithelium, and gastric parietal cells, as well as in astrocytes
and skeletal muscle plasmalemma. Because these sites correspond to membranes where orthogonal arrays of particles (OAPs) have
been observed by freeze-fracture electron microscopy, we tested the hypothesis that MIWC forms OAPs. Chinese hamster ovary
cells were stably transfected with the coding sequence of rat MIWC under a cytomegalovirus promoter. Immunostaining of clonal
cell populations showed MIWC expression at the plasma membrane. A single band at 31 kDa was detected on immunoblot. Cell fractionation
by sucrose gradient centrifugation indicated strong MIWC expression in plasma membrane fractions with lesser expression in
Golgi. Functional analysis by stopped-flow light scattering showed high mercurial insensitive water permeability in plasma
membrane vesicles. Freeze-fracture electron microscopy showed distinct OAPs on the plasma membrane P-face of MIWC-expressing
cells with morphology indistinguishable from that in basolateral membrane of kidney collecting duct; the E-face showed corresponding
linear grooves (spacing, 
8 nm) in transfected cells and collecting duct. OAPs were not observed in control (empty vector-transfected) cells or CHIP28
(AQP1)-transfected cells in which disorganized intramembrane particle aggregates were found. These results provide direct
evidence that a molecular water channel can spontaneously assemble in regular arrays.
Footnotes
-
↵* This work was supported by Grants DK35124, HL42368, DK36451, and HL51854 from the National Institutes of Health and RDP Grant R613 from the National Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- MIP
-
major intrinsic protein
- IMP
-
intramembrane particle
- OAP
-
orthogonal arrays of particles
- MIWC
-
mercurial insensitive water channel
- CHO
-
Chinese hamster ovary
- PBS
-
phosphate-buffered saline.
-
- Received December 8, 1995.
- Revision received January 8, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
