Determination of sites in the Na+H+ exchanger isoform 1 involved in cation translocation

Na+/H+ exchangers (NHEs) are a family of highly regulated, integral membrane (glyco)phosphoproteins that regulate intracellular pH and cell volume homeostasis. Compounds such as amiloride are known to be competitive antagonists of Na+ binding and previous structural studies have demonstrated that these antagonists interact with amino acids located in transmembrane domains 4 and 9 (TM4 and TM9). Wt therefore wished to test the hypothesis that TM4 and TM9 are also part of the cation translocation pore. To this end, cysteine-scanning mutagenesis was used to analyze these regions of the protein. Using a functional cysteine-less NHE1 mutant as a template, cysteine residues were introduced at unique sites in TM4 (between amino acids 166-179), TM9 (342-359) and the putative re-entrant loop (R-loop) between TM9 and TM10 (370-437). These residues were modified using methanethiosulfonate (MTS) derivatives, thiolspecific modifying reagents that will covalently bind to freely accessible cysteine residues. MTS modification of an introduced cysteine residue that faces the aqueous pore should inhibit cation translocation. As expected, these reagents did not inhibit cation exchange through the cysteine-less form of NHE1. MTS-modification of single-cysteine mutants located within TM4 had little effect on cation translocation. By contrast, three mutants located in TM9, H353C, S355C, and G356C, all confer inhibition (45-99%) in the presence of MTSET, a positively-charged MTS derivative. Furthermore, mutant S405C, located in the R-loop/TMlO, is inhibited by almost 70% in the presence of MTSET. Comparable results were also obtained with the negatively-charged derivative MTSES, although the inhibition was less pronounced. With the exception of P171C, the kinetic profiles of selected mutants in the absence of the MTS reagents remained similar to that of the wild type and cysteine-less versions of the exchanger. Our results therefore indicate a role for TM9 and the adjacent R-loop/TMlO in cation translocation