title: Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters.
abstract: Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K(+) channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K(+) channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K(+) uniporter, Arabidopsis AtHKT1-mediated Na(+) uniporter and wheat TaHKT1-mediated K(+)/Na(+) symporter) by examining K(+) uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K(+) transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K(+) channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2(D) and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.
authors.affiliation: Bioscience and Biotechnology Center, Nagoya University, Nagoya, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kato
authors.forename: Masaro
authors.initials: M
authors.lastname: Akai
authors.forename: Lalu
authors.initials: L
authors.lastname: Zulkifli
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Matsuda
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kato
authors.forename: Shinobu
authors.initials: S
authors.lastname: Goshima
authors.forename: Akihiro
authors.initials: A
authors.lastname: Hazama
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Yamagami
authors.forename: H Robert
authors.initials: HR
authors.lastname: Guy
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Uozumi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D018014:Gene Transfer Techniques; D008564:Membrane Potentials; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014908:Triticum / Q000378:metabolism*; D014982:Xenopus laevis; D015003:Yeasts / Q000254:growth & development / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C490286:HKT1 protein, Arabidopsis; C088864:HKT1 protein, plant; C113206:KtrB protein, Bacteria; D010940:Plant Proteins; D027981:Symporters; D012964:Sodium; D011188:Potassium