Objectives: The Na⁺/H⁺ exchanger NHE1 is essential to normal cell function in vertebrates. We recently presented functional evidence that residues in transmembrane (TM) domain IV and TMX-XI are important for inhibitor binding and ion transport by human (h)NHE1 (1). Here, we present a structural model of the TM region of hNHE1 that further supports this conclusion. Methods: A homology model of NHE1 was created based on the crystal structure of the E. coli Na⁺/H⁺ exchanger NhaA, taking into account experimentally identified constraints. The model was validated by site- directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy in conjunction with functional studies of hNHE1 mutants. Results: Ala173Cys (TMIV) and/or Ile461Cys (TMXI) mutations were introduced into a Cys-less hNHE1. The mutants exhibited NHE function in mammalian cells, suggesting unperturbed structures. The constructs were MTS-SL-labeled and the distance between the TMIV and TMXI spin-labels determined by EPR at about 17 Å, confirming the predicted close proximity of these domains. Reduction of pH to 5.1 or addition of the NHE1 inhibitor cariporide induced further dipolar broadening corresponding to a decrease in distance to ~13 Å. Also in accordance with the model a teleost NHE1, paNHE1, which is insensitive to cariporide (1) showed a similar EPR response to pH 5.1, yet no response to cariporide. The model predicts that Arg425 in TMX could stabilize an energetically unfavorable dipole-dipole arrangement in the TMIV/XI crossover. Accordingly, an Arg425Ala mutation resulted in failure of hNHE1 membrane targeting in mammalian cells and reduced NHE1 function. Conclusion: We have created and experimentally validated a homology model of hNHE1, and data are consistent with a role for TMIV-TMXI rearrangement in ion translocation and inhibitor binding by hNHE1. Additional EPR analyses are ongoing to further refine the model. 1. Pedersen, S.F. et al. J Biol Chem 282: 19716- 19727, 2007