Abstract
Previously, the only known blockers of water permeability through aquaporin-1 (AQP1) water channels were mercurial reagents such as HgCl2. For AQP1, inhibition by mercury has been attributed to the formation of a mercaptide bond with cysteine residue 189 found in the putative pore-forming region loop E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed inXenopus oocytes. After preincubation of the oocytes for 15 min with 100 μM TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of partial block similar to that obtained with 15 min of incubation in 100 μM HgCl2. The reduction of water permeability was dose-dependent for tested concentrations up to 10 mM TEA. TEA blocks the Shaker potassium channel by interacting with a tyrosine residue in the outer pore region. We tested whether an analogous tyrosine residue in loop E of AQP1 could be involved in the binding of TEA. Using polymerase chain reaction, tyrosine 186 in AQP1, selected for its proximity to the mercury-binding site, was mutated to phenylalanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression of the mutant AQP1 channels showed that the water permeability of Y186F was equivalent to that of wild-type AQP1; the other mutant channels did not conduct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 μM TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E.
Footnotes
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Send reprint requests to: Andrea J. Yool, Ph.D., Department of Physiology, P.O. Box 24-5051, University of Arizona College of Medicine, Tucson, AZ 85724. E-mail:ayool{at}u.arizona.edu
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↵1 Current address: Laboratory of Kidney and Electrolyte Metabolism, National Institutes of Health, Bldg. 10, Rm. 6N240, 10 Center Dr., MSC 1603, Bethesda, MD 20892-1603.
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This work was supported by American Heart Association Desert/Mountain Affiliate 9951130Z and National Institutes of Health Grant EY11291. This work was presented previously in abstract form (FASEB J 1999, 13:A394).
- Abbreviations:
- AQP1
- aquaporin-1
- TEA
- tetraethylammonium
- TMA
- tetramethylammonium
- TPA
- tetrapropylammonium
- MIP
- membrane intrinsic protein
- RVI
- relative volume increase
- Pf
- osmotic permeability
- PCR
- polymerase chain reaction
- Received July 26, 1999.
- Accepted January 6, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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