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, Peter M Cala

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)19716-19727
Number of pages12
JournalJournal of Biological Chemistry
Volume282
Issue number27
DOIs
StatePublished - Jul 6 2007

Fingerprint

Amiloride
Holographic optical elements
Tail
Flounder
Urodela
Sodium-Hydrogen Antiporter

ASJC Scopus subject areas

  • Biochemistry

Cite this

NHE1 inhibition by amiloride- and benzoylguanidine-type compounds : Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity. / Pedersen, Stine F.; King, Scott A.; Nygaard, Eva B.; Rigor, Robert R.; Cala, Peter M.

In: Journal of Biological Chemistry, Vol. 282, No. 27, 06.07.2007, p. 19716-19727.

Research output: Contribution to journalArticle

@article{577e3a5c28d847c892b761fceb5439b5,
title = "NHE1 inhibition by amiloride- and benzoylguanidine-type compounds: Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity",
abstract = "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.",
author = "Pedersen, {Stine F.} and King, {Scott A.} and Nygaard, {Eva B.} and Rigor, {Robert R.} and Cala, {Peter M}",
year = "2007",
month = "7",
day = "6",
doi = "10.1074/jbc.M701637200",
language = "English (US)",
volume = "282",
pages = "19716--19727",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "27",

}

TY - JOUR

T1 - NHE1 inhibition by amiloride- and benzoylguanidine-type compounds

T2 - Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity

AU - Pedersen, Stine F.

AU - King, Scott A.

AU - Nygaard, Eva B.

AU - Rigor, Robert R.

AU - Cala, Peter M

PY - 2007/7/6

Y1 - 2007/7/6

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=34547115979&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34547115979&partnerID=8YFLogxK

U2 - 10.1074/jbc.M701637200

DO - 10.1074/jbc.M701637200

M3 - Article

C2 - 17493937

AN - SCOPUS:34547115979

VL - 282

SP - 19716

EP - 19727

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 27

ER -