Structural and Functional Characterization of Transmembrane Segment VII of the Na+/H+ Exchanger Isoform 1

Abstract

The Na؉ /H ؉ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH by exchanging one intracellular H ؉ for one extracellular Na ؉ . It is composed of an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the structural and functional aspects of the critical transmembrane segment VII (TM VII, residues 251-273) by using alanine scanning mutagenesis and high resolution NMR. Each residue of TM VII was mutated to alanine, the full-length protein expressed, and its activity characterized. TM VII was sensitive to mutation. Mutations at 13 of 22 residues resulted in severely reduced activity, whereas other mutants exhibited varying degrees of decreases in activity. The impaired activities sometimes resulted from low expression and/or low surface targeting. Three of the alanine scanning mutant proteins displayed increased, and two displayed decreased resistance to the Na ؉ /H ؉ exchanger isoform 1 inhibitor EMD87580. The structure of a peptide of TM VII was determined by using high resolution NMR in dodecylphosphocholine micelles. TM VII is predominantly ␣-helical, with a break in the helix at the functionally critical residues Gly 261 -Glu 262 . The relative positions and orientations of the N-and C-terminal helical segments are seen to vary about this extended segment in the ensemble of NMR structures. Our results show that TM VII is a critical transmembrane segment structured as an interrupted helix, with several residues that are essential to both protein function and sensitivity to inhibition. The mammalian Naϩ /H ϩ exchanger isoform 1 (NHE1) 6 is a ubiquitous integral membrane protein mediating removal of a single intracellular proton in exchange for one extracellular sodium ion (1). NHE1 has several cellular and physiological functions, including protecting cells from intracellular acidification (2, 3), promoting cell growth and differentiation (2), and regulating sodium fluxes and cell volume after osmotic shrinkage (4). The Na ϩ /H ϩ exchanger also plays a critical role in the damage that occurs during ischemia and reperfusion and may play a key role in mediating heart hypertrophy. Inhibition of the exchanger protects the myocardium in these two forms of heart disease (5, 6). Amiloride and its derivatives are inhibitors of the NHE1 isoform of the Na ϩ /H ϩ exchanger, and a new generation of Na ϩ /H ϩ exchanger inhibitors is being developed for clinical treatment of heart disease (7,8). In addition to these many physiological roles, more recently, the Na ϩ /H ϩ exchanger has been demonstrated to be involved in modulating cell motility and invasiveness of neoplastic breast cancer cells (9) and has been shown to be critical to cell motility in some cell types (10).NHE1 is composed of two domains as follows: an N-terminal membrane domain of ϳ500 amino acids and a C-terminal regulatory domain of ϳ315 amino acids (1, 6). The N-terminal membrane domain is responsible for ion movement and has 12 transmembrane segments ...

Dimeric Structure of Human Na+/H+ Exchanger Isoform 1 Overproduced in Saccharomyces cerevisiae

“…The measured IC 50 value for hNHE1 inhibition by EMD87580 was 5 M (Fig. 4C), which is similar to the value reported for wild-type hNHE1 expressed in mammalian cells (39). Fig.…”

“…The addition of (NH 4 ) 2 SO 4 completely collapsed the cation gradient across the vesicle membranes and resulted in full recovery of pyranine fluorescence. When vesicles were preincubated with the amiloride analog EMD87580, a potent specific NHE1 inhibitor (39), cation exchange activity of the hNHE1 proteoliposomes was inhibited in a dose-dependent manner (Fig. 4B).…”

Model Structure of the Na+/H+ Exchanger 1 (NHE1)

“…3). Extensive mutagenesis within TM5 in NHE1 demonstrated its importance for expression and targeting to the membrane (30), in accordance with its strategic location in the protein core in our model.…”

“…Some of these mutations do not affect Na ϩ affinity, implying that the inhibitorbinding site is physically distinct and suggesting that the inhibitors induce allosteric regulation (30). FIGURE 4.…”

“…Asp 267 is located at the bottom of the cytoplasmic funnel (Fig. 1B) and, by conjecture, is involved in cation binding; a negative charge at this position is indeed crucial for function (30), and even a mild substitution abolishes the activities of NHE1 and its yeast homologue sod2 (Tables 1S and 2S (Table 1S), and facing adjacent helices, we suggest that it might be of structural importance. Further mutations of these residues in both EcNhaA and NHE1 are likely to shed some light on their role in determining the Na ϩ :H ϩ stoichiometry of the transporters.…”