title: The unwound portion dividing helix IV of NhaA undergoes a conformational change at physiological pH and lines the cation passage.
abstract: pH and Na(+) homeostasis in all cells requires Na(+)/H(+) antiporters. The crystal structure of NhaA, the main antiporter of Escherichia coli, has provided general insights into antiporter mechanisms and their pH regulation. Functional studies of NhaA in the membrane have yielded valuable information regarding its functionality in situ at physiological pH. Here, we Cys-scanned the discontinuous transmembrane segment (TM) IV (helices IVp and IVc connected by an extended chain) of NhaA to explore its functionality at physiological pH. We then tested the accessibility of the Cys replacements to the positively charged SH reagent [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and the negatively charged 2-sulfonatoethyl methanethiosulfonate (MTSES) in intact cells at pH 8.5 and 6.5 and in parallel tested their accessibility to MTSET in high-pressure membranes at both pH values. We found that the outer membrane of E. coli TA16 acts as a partially permeable barrier to MTSET. Overcoming this technical problem, we revealed that (a) Cys replacement of the most conserved residues of TM IV strongly increases the apparent K(m) of NhaA to both Na(+) and Li(+), (b) the cationic passage of NhaA at physiological pH is lined by the most conserved and functionally important residues of TM IV, and (c) a pH shift from 6.5 to 8.5 induces conformational changes in helix IVp and in the extended chain at physiological pH.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University, 91904 Jerusalem, Israel.
authors.forename: Abraham
authors.initials: A
authors.lastname: Rimon
authors.forename: Lena
authors.initials: L
authors.lastname: Kozachkov-Magrisso
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
mesh_terms: D019943:Amino Acid Substitution; D002412:Cations / Q000378:metabolism; D002463:Cell Membrane Permeability; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008698:Mesylates / Q000494:pharmacology; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D002412:Cations; D029968:Escherichia coli Proteins; D008698:Mesylates; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate