Structural and Functional Characterization of Transmembrane Segment IX of the NHE1 Isoform of the Na+/H+ Exchanger

Abstract

The Na؉ /H ؉ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by removing one intracellular H ؉ in exchange for one extracellular Na ؉ . It has a large N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the putative transmembrane segment IX (residues 339 -363). Each residue was mutated to cysteine in a functional cysteineless NHE1 protein. Of 25 amino acids mutated, 5 were inactive or nearly so after mutation to cysteine. Several of these showed aberrant targeting to the plasma membrane and reduced expression of the intact protein, whereas others were expressed and targeted correctly but had defective NHE1 The mammalian Na ϩ /H ϩ exchanger isoform 1 (NHE1) 6 is a ubiquitous integral membrane protein that regulates intracellular pH by mediating removal of one intracellular proton in exchange for one extracellular sodium ion (1). NHE1 has many cellular and physiological functions. It promotes cell growth and differentiation (2), protects cells from intracellular acidification (2, 3), and also regulates sodium fluxes and cell volume after challenge by osmotic shrinkage (4). In addition, Na ϩ /H ϩ exchanger activity has been demonstrated to be involved in modulating cell motility and invasiveness of neoplastic breast cancer cells (5), and it has been shown to be critical to cell motility in some cell types (6). The Na ϩ /H ϩ exchanger also plays a critical causal role in heart hypertrophy and in the damage that occurs during ischemia and reperfusion. Inhibition of the exchanger with a new generation of Na ϩ /H ϩ exchanger inhibitors protects the myocardium during these disease states (7-10).NHE1 has an N-terminal membrane ion translocation domain of ϳ500 amino acids and a C-terminal regulatory domain of ϳ315 amino acids (1, 8). Wakabayashi et al. (11) used a combination of hydrophobicity analysis and cysteine-scanning mutagenesis to predict a topology with 3 membrane-associated segments and 12 integral transmembrane (TM) segments (Fig. 1A). A recent homology model, based on the Escherichia coli antiporter NhaA crystal structure (12), also predicts 12 TM segments, but assignments of transmembrane segments are different in comparison with the Wakabayashi topology (13).The exact details of cation coordination and translocation through the central ion pore of NHE1 have only recently begun to emerge. In this respect, we have identified TM IV (14) and TM VII (15) (with topology assignments according to Ref. 11) as functionally critical. More recently, we solved the structures of these TM segments and identified specific essential residues (16,17). Prolines 167 and 168 of TM IV were essential to NHE1 function (14) and Phe 161 was shown to be a porelining residue involved in transport using cysteine-scanning mutagenesis (16). The peptide was composed of an N-terminal ␤-turn region, followed by an extended (nonhelical) structure * This work was supported in part by gr...

Structure and function of the human Na+/H+exchanger isoform 1

“…Their structure is more similar to that of part of TM 7 and 8 with a link in between. In addition, the same study 35 showed that Ser 351 is a pore lining residue which is in agreement with model 2's predictions.…”

“…Additionally, there have been nuclear magnetic resonance (NMR) studies on individual putative TM helices of NHE1 taken from the Wakabayashi model. The solution NMR structures have been elucidated for the putative TM segments IV, VII and IX [33][34][35] while that of TM XI has also been determined (unpublished observation). This detailed structural information allows more informed functional assays as well as the prediction of the molecular mechanism of NHE1 function.…”

“…The structure of amino acids 339-363, TM IX of model 1, was that of a kinked peptide with helices at either end. 35 These amino acids were predicted to be TM IX in model 1, but were predicted to be part of TM segments 7 and 8 of model 2. Their structure is more similar to that of part of TM 7 and 8 with a link in between.…”

Structural and Functional Analysis of Transmembrane Segment IV of the Salt Tolerance Protein Sod2*

“…This was done to aid solubilization of the peptide as described earlier (13,15). The primers SodMBPf (5Ј-CATGGGATCCAAAAAAAAATTGT-TTCCACAAATTAACTTTTTAGG-3Ј) and SodMBPr (5Ј-CCGGGAATTCTCATTTCTTTTTTCCTACAATCAATG-CTGATAG-3Ј) were used to amplify the DNA of sod2 and add the terminal lysines.…”

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

“…TM IX contains two structurally conserved regions containing ␣-helix structure at residues 340 -344 and 353-359, with a 90°kink at Ser 351 between the two regions that could potentially provide flexibility. Cysteine scanning mutagenesis showed that both residues Ser 351 and Glu 346 , between the helical regions, are pore-lining (18). TM XI contains two helical regions, residues 447-454 and 460 -471, and Leu 465 was identified as pore-accessible through cysteine scanning mutagenesis and reaction with MTSET (19).…”