Freeze-Fracture Analysis of Plasma Membranes of CHO Cells Stably Expressing Aquaporins 1-5 |
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Authors: | AN van Hoek B Yang S Kirmiz D Brown |
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Institution: | (1) Renal Unit and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA, US;(2) Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA, US |
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Abstract: | Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy.
For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1–5. Measurement of the
osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane.
By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes
of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected
cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied
10–20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters
were detected in ∼5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs
showed a significant shift from 13.5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis
of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells
had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected
CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only
AQP4 assembles into well-defined IMP arrays.
Received: 17 March 1998/Revised: 19 June 1998 |
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Keywords: | : Freeze fracture — Orthogonal square array — Intramembrane particle — Aquaporin — Chinese Hamster ovary cell — Transfection |
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