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

doi:10.1074/jbc.m701637200

Stine F Pedersen, Scott A King + Show 3 more

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https://doi.org/10.1074/jbc.m701637200

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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.

Highlights

The ubiquitous plasma membrane Naϩ/Hϩ exchanger, NHE1, plays a major role in the regulation of cellular pH, cell volume, and cytoskeletal organization in most cell types, and it has been implicated in the control of a wide variety of cellular functions, including migration, proliferation, and cell death [1,2,3,4]
We confirmed the important role of TM4 in determining the inhibitor sensitivity of NHE1, and we demonstrated for the first time that regions in TM10 –11 and/or the associated intra- and extracellular loops (IL5 and EL6) play a major role in NHE1 inhibitor sensitivity
The AtNHE1 in native RBCs was inhibited by the 5Ј-N-substituted amiloride derivative EIPA (22Naϩ influx measurements),3 whereas EIPA was without effect on PaNHE1 in RBCs in concentrations of up to 100 ␮M (n ϭ 3 at 20 ␮M, and n ϭ 3 at 100 ␮M; data not shown)

Summary

Introduction

The ubiquitous plasma membrane Naϩ/Hϩ exchanger, NHE1, plays a major role in the regulation of cellular pH, cell volume, and cytoskeletal organization in most cell types, and it has been implicated in the control of a wide variety of cellular functions, including migration, proliferation, and cell death [1,2,3,4]. The two smaller loops are located intracellularly between TM4 and -5 (i.e. intracellular loop (IL) 2) and TM8 and -9 (i.e. IL4), respectively, whereas the large loop, the fifth extracellular loop (EL5), which resembles the poreforming loops found in voltage-gated ion channels, has an extracellular origin and presumably inserts into the exofacial surface between TM9 and -10 [2, 14]. These putative re-entrant regions are highly conserved among hNHE1, AtNHE1, 19716 JOURNAL OF BIOLOGICAL CHEMISTRY. The apparent major role of TM9 was supported by the findings that replacement of 66 amino acids in and around TM9 of NHE1 with the corresponding residues of NHE3 decreased inhibitor sensitivity [21], and that targeted replacement of Glu350 (rat)/Glu346 (human) or Gly356 (rat) in TM9 reduced sensitivity to both amiloride- and HOE-type compounds [20, 22]

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