title: The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport.
abstract: Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kuwabara
authors.forename: Hiroki
authors.initials: H
authors.lastname: Inoue
authors.forename: Yumi
authors.initials: Y
authors.lastname: Tsuboi
authors.forename: Norihiro
authors.initials: N
authors.lastname: Nakamura
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kanazawa
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology; D016480:Helicobacter pylori / Q000378:metabolism*; D017136:Ion Transport; D007477:Ions; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008094:Lithium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D012694:Serine / Q000737:chemistry; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000502:physiology; D013912:Threonine / Q000737:chemistry; D014633:Valine / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: D017920:Antiporters; D029968:Escherichia coli Proteins; D007477:Ions; C102839:NhaA protein, E coli; D011522:Protons; D017923:Sodium-Hydrogen Exchangers; D014867:Water; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D010649:Phenylalanine; D004247:DNA; D008094:Lithium; D007930:Leucine; D014633:Valine; D003545:Cysteine; D005033:Ethylmaleimide; D000409:Alanine