Inhibition of aquaporin-1 water permeability by tetraethylammonium: Involvement of the loop E pore region

Previously, the only known blockers of water permeability through aquaporin -1 (AQP1) water channels were mercurial reagents such as HgCl2. For AQP1, i nhibition by mercury has been attributed to the formation of a mercaptide b ond with cysteine residue 189 found in the putative pore-forming region loo p E. Here we show that the nonmercurial compound, tetraethylammonium (TEA) chloride, reduces the water permeability of human AQP1 channels expressed i n Xenopus oocytes. After preincubation of the oocytes for 15 min with 100 m u M TEA, AQP1 water permeability was reduced by 20 to 40%, a degree of part ial block similar to that obtained with 15 min of incubation in 100 mu M Hg Cl2. The reduction of water permeability was dose-dependent for tested conc entrations up to 10 mu M TEA. TEA blocks the Shaker potassium channel by in teracting with a tyrosine residue in the outer pore region. We tested wheth er 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, sel ected for its proximity to the mercury-binding site, was mutated to phenyla lanine (Y186F), alanine (Y186A), or asparagine (Y186N). Oocyte expression o f 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 con duct water. However, in contrast to wild-type AQP1, the water permeability of Y186F was not reduced with 100 mu M TEA. These results suggest that TEA reduces AQP1 water permeability by interacting with loop E.