Leu143 in the Putative Fourth Membrane Spanning Domain Is Critical for Amiloride Inhibition of an Epithelial Na+/H+ Exchanger Isoform (NHE-2)

Yun, Chc, Little, PJ, Nath, SK, Levine, SA, Pouyssegur, J, Tse, CM and Donowitz, M (1993) Leu143 in the Putative Fourth Membrane Spanning Domain Is Critical for Amiloride Inhibition of an Epithelial Na+/H+ Exchanger Isoform (NHE-2). Biochemical and Biophysical Research Communications, 193 2: 532-539. doi:10.1006/bbrc.1993.1656


Author Yun, Chc
Little, PJ
Nath, SK
Levine, SA
Pouyssegur, J
Tse, CM
Donowitz, M
Title Leu143 in the Putative Fourth Membrane Spanning Domain Is Critical for Amiloride Inhibition of an Epithelial Na+/H+ Exchanger Isoform (NHE-2)
Journal name Biochemical and Biophysical Research Communications   Check publisher's open access policy
ISSN 0006-291X
Publication date 1993-06-15
Sub-type Article (original research)
DOI 10.1006/bbrc.1993.1656
Volume 193
Issue 2
Start page 532
End page 539
Total pages 8
Language eng
Subject 1312 Molecular Biology
1304 Biophysics
1303 Specialist Studies in Education
Abstract A family of Na+/H+ exchanger isoforms (called NHE1, NHE2, and NHE3) which exhibits a wide range of amiloride sensitivity has recently been cloned and characterized. A part of the domain, which determines amiloride sensitivity in the epithelial Na+/H+ exchanger isoform, NHE2, was identified by site-directed mutagenesis and functional studies using cDNAs stably expressed in a fibroblast cell line. It has previously been reported that AR300, an amiloride resistant mutant of the ubiquitous Na+/H+ exchanger isoform, NHE1, is 30-fold more resistant to methylpropyl amiloride (MPA) compared to NHE1 and contains a single amino acid substitution of L167F in the fourth putative transmembrane helix, which corresponds to L143 in NHE2. Therefore, in the present study point mutational substitutions were introduced into the equivalent of this fourth transmembrane helix of rabbit NHE2 (including Y144F; L143F; L143F and Y144F) to mimic the corresponding amino acids in NHE1, NHE3 (another epithelial isoform) and AR300, respectively. NHE2/L143F (mimicking NHE3) increased the IC50 for amiloride by 5-fold and for ethylisopropyl amiloride (EIPA) by 20-fold. Similarly, NHE2/L143F and Y144F (mimicking AR300) increased the resistance to both amiloride and EIPA by 10-fold. On the other hand, NHE2/Y144F (mimicking NHE1) did not affect the sensitivity to amiloride or EIPA, and this mutant, like wild type NHE2, is partially resistant to EIPA. Thus, amino acid 143 of NHE2 is critical for, but is not the only amino acid responsible for, amiloride and EIPA inhibition of Na+/H+ exchange. That none of the mutations studied altered the Na+ affinity of these Na+/H+ exchangers further suggests that amiloride binding and Na+ transport sites are not identical.
Q-Index Code C1
Q-Index Status Provisional Code
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Sub-type: Article (original research)
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