The short C-terminal hydrophilic domain of NhaH Na^＋/H^＋ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na^＋/H^＋ antiporter plays key roles in maintaining low cytoplasmic NaNa^＋ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na^＋/H^＋ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues （^395PLIKKLGMI403） existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaH△C. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coil KNabc, in which three main Na^＋/H^＋ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coil KNabc/nhaHAC decreased both Na^＋/H^＋ and Li^＋/H^＋ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na^＋, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing.

The short C-terminal hydrophilic domain of NhaH Na+/H+ antiporter from Halobacillus dabanensis with roles in resistance to salt and in pH sensing

The Na⁺/H⁺ antiporter plays key roles in maintaining low cytoplasmic Na⁺ level and pH homeostasis, while little is known about the Carboxyl-terminal hydrophilic tails of prokaryotic antiporters. In our previous study, the first Na⁺/H⁺ antiporter gene nhaH from moderate halophiles was cloned from Halobacillus dabanensis D-8 by functional complementation. A topological model suggested that only nine amino acid residues (³⁹⁵PLIKKLGMI⁴⁰³) existed in the hydrophilic C-terminal domain of NhaH. The C-terminal truncated mutant of NhaH was constructed by PCR strategy and designated as nhaHΔC. Salt tolerance experiment demonstrated that the deletion of hydrophilic C-terminal nine amino acid residues significantly inhibited the complementation ability of E. coli KNabc, in which three main Na⁺/H⁺ antiporters nhaA, nhaB and chaA were deleted. Everted membrane vesicles prepared from E. coli KNabc/nhaHΔC decreased both Na⁺/H⁺ and Li⁺/H⁺ exchange activities of NhaH, and also resulted in an acidic shift of its pH profile for Na⁺, indicating a critical role of the short C-terminal domain of NhaH antiporter in alkali cation binding/translocation and pH sensing. Supported by the National High Technology Research and Development Program of China (Grant No. 2003AA241150), and International Cooperation Program for Science and Technology (Grant No. 2006DFA31060).