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NHE1 inhibition by amiloride‐ and benzoyl guanidine‐type inhibitors: inhibitor binding loci deduced from chimeras of three NHE1 homologs with markedly different inhibitor sensitivity
Author(s) -
Nygaard Eva,
King Scott A.,
Rigor Robert R.,
Cala Peter M.,
Pedersen Stine F.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a964-a
Subject(s) - amiloride , chemistry , guanidine , mutant , microbiology and biotechnology , biochemistry , biology , sodium , gene , organic chemistry
The interaction of the Na + /H + exchanger, NHE1, with amiloride and benzoyl‐guanidine (HOE)‐type compounds is incompletely understood. We previously cloned NHE1 homologs from A. tridactylum (atNHE1) and P. americanus (paNHE1). In spite of high homology to the amiloride‐, EIPA‐, and HOE‐sensitive human NHE1 (hNHE1), atNHE is inhibitable by amiloride‐type yet insensitive to HOE‐type compounds, and paNHE is insensitive to all these compounds. Here, we employed a comparative approach to identify NHE1 regions responsible for inhibitor interaction. Replacing the C‐terminal tail of paNHE1 with that of atNHE1 had no effect on amiloride sensitivity. However, amiloride‐ (IC 50 249 ± 2 μM), yet not HOE642‐sensitivity was conferred to paNHE1 by replacing TM10‐11 with those of atNHE1. In contrast, the converse mutant (atNHE1 TM10‐11 replaced with that of paNHE1), was HOE642 sensitive. Replacing a LFFFY motif in TM4 of paNHE1 with the corresponding residues (VFFLF) in hNHE1 conferred sensitivity to amiloride (IC 50 277 ± 2 μM), EIPA (IC 50 9 ± 2.8 μM), and HOE694. Replacing the LFFFY motif with the corresponding motif (TFFLF) in atNHE1 also conferred HOE694 sensitivity although atNHE1 is HOE694‐insensitive. Thus, regions in TM4 and TM10‐11 can independently confer amiloride‐ and EIPA sensitivity to paNHE1. Moreover, TM4 is necessary, yet not sufficient, for HOE694 sensitivity, which is also dependent on regions in TM10‐11.