Functional analysis of the M2(D) helix of the TRK1 potassium transporter of Saccharomyces cerevisiae.
Eukaryotic KcsA-related K+ transporters mediate physiologically relevant K+ and Na+ fluxes in fungi and plants. ScTRK1 is a characteristic member of the group, and here we report a mutational analysis of the unique M2(D) helix of this transporter. Our results support the theoretical models placing this helix in a relevant position in the pore and interacting with P segments. Most single mutations eliminating positively charged or introducing negatively charged residues reduced the V(max) of Rb+ influx to a half, several together showed an additive effect, and four practically suppressed transport. In contrast, the introduction of only one positively charged residue practically abolished the function of the transporter. Almost all mutations in the M2(D) helix affected the two Rb+ binding sites of the transporter, mimicking mutations in the selectivity filter.
Haro
Rosario
R
Departamento de Biotecnología, Laboratorio de Microbiología, Universidad Politécnica de Madrid, Escuela Técnica Superior de Ingenieros Agrónomos, 28040 Madrid, Spain.
Rodríguez-Navarro
Alonso
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012413:Rubidium / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment
D027682:Cation Transport Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C068664:TRK1 protein, S cerevisiae; D012413:Rubidium