Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na+/H+ antiporter of Halobacillus dabanensis

AbstractNhaH is a novel Na+/H+ antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative transmembrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na+(Li+)/H+ antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na+ and Li+ without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na+ and Li+ but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H+ binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na+ and Li+, indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li+, without affecting that for Na+, suggesting that E229 may be mainly responsible for Li+ binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na+ and Li+, indicating that H87 and H88 are involved in H+ binding or pH regulation