Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1

Abstract

We previously presented evidence that transmembrane domain (TM) IV and TM X-XI are important for inhibitor binding and ion transport by the human Na The ubiquitous plasma membrane Na ϩ /H ϩ exchanger isoform 1 (NHE1) 4 plays central roles in cellular pH and volume homeostasis, cell migration, proliferation, and survival, and increased NHE1 activity contributes to ischemia-reperfusion injury as well as tumor growth and proliferation (1, 2). Hence, the ability to selectively block NHE1, although of high clinical relevance, is hampered by a lack of detailed understanding of NHE1 structure and mechanism of ion translocation. Hydropathy analyses and accessibility studies indicate that NHE1 has 12 transmembrane (TM) segments and a large Cterminal cytoplasmic region (3). Cysteine accessibility studies suggest the presence of two small re-entrant loops between TM IV and TM V (intracellular loop (IL) II) and TM VIII and TM IX (IL IV), respectively, and a larger re-entrant loop between TM IX and TM X (extracellular loop V) (1, 3). Portions of IL II and IL IV are located within the membrane and accessible from either side of the membrane, suggesting that they may form structures lining an aqueous pore and could be involved in ion translocation by NHE1 (3). Extracellular loop V is also interesting in this regard, because it resembles the Ploops found in voltage-gated ion channels (1, 3). These putative re-entrant loops are highly conserved among several NHE1 homologs, consistent with the notion that they are critical for NHE1 function (3-5).A number of regions within the NHE1 protein have been implicated in inhibitor binding, e.g. TM IV and TM IX (6 -11); however, the mechanism(s) of interaction between NHE1 and its commonly used inhibitors, amiloride and benzoyl guanidine type compounds, remain to be fully elucidated.Using a comparative approach based on chimeras generated using human NHE1 (hNHE1) and two NHE1 homologs (flounder paNHE1 and Amphiuma tridactylum NHE1) with high sequence homology to hNHE1 yet markedly different inhibitor profiles (4, 5), we previously obtained novel information on the regions of NHE1 important for inhibitor binding and ion transport (12). These studies confirmed that TM IV plays a central role in inhibitor binding (12) as suggested by earlier point mutation studies (6 -11). Moreover, we demonstrated that regions in TM X-XI and/or IL V and extracellular loop VI are important determinants of inhibitor sensitivity (12).The three-dimensional structure of NHE1 is unknown; however, the structure of the distantly related bacterial (Esch-

Na+/H+ Exchange and Hypoxic Pulmonary Hypertension

“…Fibroblasts that express an NHE1 variant with a mutation in the ezrin-binding motif have impaired organization of actin stress fibers and an irregular cell shape, while fibroblasts expressing an NHE1 mutant with disrupted ion translocation function have normal cytoskeletal organization and cell shape. 33 Furthermore, fibroblasts containing NHE1 with mutations in the C-terminal ezrin-binding motif also show evidence of impaired cell migration. 69 As is implied in the name, mutations in the C-terminal ezrinbinding motif impair the ability of NHE1 to bind with ezrin, 33 a member of a family of proteins referred to as ERM (ezrin, radixin, moesin) proteins.…”

“…33 Furthermore, fibroblasts containing NHE1 with mutations in the C-terminal ezrin-binding motif also show evidence of impaired cell migration. 69 As is implied in the name, mutations in the C-terminal ezrinbinding motif impair the ability of NHE1 to bind with ezrin, 33 a member of a family of proteins referred to as ERM (ezrin, radixin, moesin) proteins. 144 ERM proteins are characterized by their shared N-terminal FERM (4.1, ezrin, radixin, moesin) domain, which binds to various membrane proteins, and a shared C-terminal filamentous-actin binding site.…”

“…Model 1 represents the structure predicted by Landau et al 30 and is based on the crystal structure of Escherichia coli NHaA. Model 2, predicted by Nygaard et al, 33 is based on E. coli NhaA, experimental models of NHE1, and electron paramagnetic resonance spectroscopy. B, Predicted topology of NHE1.…”

“…In another model, TM domains 4 and 11 form a central core. 33 Binding of H + ions at TM9 causes a conformational change and direct contact between TM4 and TM9. As a result, TM4 and TM11 are reoriented, exposing a Na + -binding site to the extracellular space.…”

“…Release of the Na + ion allows protonation of the Na + -binding site and rearrangement back to the original conformation. 33 The cytosolic C-terminal domain of NHE1 contains a number of sites important for phosphorylation and interaction with regulatory molecules. For example, calmodulin, a protein whose activation is dependent on intracellular Ca 2+ levels, binds at amino acids 636-656.…”