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J. Biol. Chem., Vol. 282, Issue 27, 19716-19727, July 6, 2007

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NHE1 Inhibition by Amiloride- and Benzoylguanidine-type Compounds

INHIBITOR BINDING LOCI DEDUCED FROM CHIMERAS OF NHE1 HOMOLOGUES WITH ENDOGENOUS DIFFERENCES IN INHIBITOR SENSITIVITY^*

Stine F. Pedersen, Scott A. King, Eva B. Nygaard, Robert R. Rigor, and Peter M. Cala¹

From the Department of Physiology and Membrane Biology, University of California, Davis, California 95616 and the Department of Molecular Biology, University of Copenhagen, DK-2100 Copenhagen, Denmark

The interaction of the ubiquitous Na⁺/H⁺ exchanger, NHE1, with its commonly used inhibitors, amiloride- and benzoylguanidine (Hoechst type inhibitor (HOE))-type compounds, is incompletely understood. We previously cloned NHE1 from Amphiuma tridactylum (AtNHE1) and Pleuronectes americanus (PaNHE1). Although highly homologous to the amiloride- and HOE-sensitive human NHE1 (hNHE1), AtNHE1 is insensitive to HOE-type and PaNHE1 to both amiloride- and HOE-type compounds. Here we generated chimeras to "knock in" amiloride and HOE sensitivity to PaNHE1, and we thereby identified several NHE1 regions involved in inhibitor interaction. The markedly different inhibitor sensitivities of hNHE1, AtNHE1, and PaNHE1 could not be accounted for by differences in transmembrane (TM) region 9. Replacing TM10 through the C-terminal tail of PaNHE1 with the corresponding region of AtNHE1 partially restored sensitivity to amiloride and the related compound 5'-(N-ethyl-N-isopropyl)amiloride (EIPA) but not to HOE694. This effect was not due to the tail region, but it was dependent on TM10–11, because replacing only this region with that of AtNHE1 also partially restored amiloride and EIPA but not HOE sensitivity. The converse mutant (TM10–11 of AtNHE1 replaced with those of PaNHE1) exhibited even higher amiloride and EIPA sensitivity and was also HOE-sensitive. Replacing an LFFFY motif in TM region 4 of PaNHE1 with the corresponding residues of hNHE1 (VFFLF) or AtNHE1 (TFFLF) greatly increased sensitivity to both amiloride- and HOE-type compounds, despite the fact that AtNHE1 is HOE694-insensitive. Gain of amiloride sensitivity appeared to correlate with increased Na⁺/H⁺ exchange rates. It is concluded that regions within TM4 and TM10–11 contribute to amiloride and HOE sensitivity, with both regions imparting partial inhibitor sensitivity to NHE1.

Received for publication, February 23, 2007 , and in revised form, May 9, 2007.

^* This work was supported by National Institutes of Health Grant HL-21179 (to P. M. C.), a Philip Morris external research program postdoctoral fellowship (to S. A. K.), and grants from the Carlsberg Foundation and the Danish National Research Council (to S. F. P.). 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 To whom correspondence should be addressed: Dept. of Physiology and Membrane Biology, School of Medicine, University of California Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-752-1285; Fax: 530-752-5423; E-mail: pmcala{at}ucdavis.edu.

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