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J. Biol. Chem., Vol. 281, Issue 20, 14207-14214, May 19, 2006

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Quaternary Ammonium Compounds as Water Channel Blockers

SPECIFICITY, POTENCY, AND SITE OF ACTION^*

Frank J. M. Detmers, Bert L. de Groot, E. Matthias Müller, Andrew Hinton^¶, Irene B. M. Konings, Mozes Sze, Sabine L. Flitsch^¶1, Helmut Grubmüller, and Peter M. T. Deen²

From the Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, Nijmegen, The Netherlands, the Max-Planck Institute for Biophysical Chemistry, Theoretical and Computational Biophysics Department, Am Fassberg, D-37077, Göttingen, Germany, and the ^¶Department of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, EH9 3JJ Scotland, United Kingdom

Excessive water uptake through Aquaporins (AQP) can be life-threatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC₅₀ values of 1.4, 6.2, and 9.8 µM, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp¹²⁸) and E (Asp¹⁸⁵) loop, and the A(Tyr³⁷-Asn⁴²-Thr⁴⁴) loop of the neighboring monomer, but not directly with Tyr¹⁸⁶. The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound.

Received for publication, December 7, 2005 , and in revised form, March 16, 2006.

^* This work was supported by European Union Grants QLRT-2000-00778 (to S. L. F., H. G., and P. M. T. D.) and QLK3-CT-2001-00987 (to H. G. and P. M. T. D.) and a grant from the BBSRC (to S. L. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ School of Chemistry and Manchester Interdisciplinary Biocentre, 131 Princess St., Manchester M1 7ND, UK.

² To whom correspondence should be addressed: 286, Dept. of Physiology, RUNMC Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-243617347; Fax: 31-243616413; E-mail: p.deen{at}ncmls.ru.nl.

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