title: Identification of key residues in the formate channel FocA that control import and export of formate.
abstract: The formate-nitrite transporter (FNT) family comprises pentameric channels that transport monovalent anions. The prototype of this family is the formate channel (FocA), which was originally identified as a formate channel in Escherichia coli. Each protomer in the channel has a pore with structural features that include periplasmic and cytoplasmic constriction sites, which are likely important for bi-directional gating of substrate passage. Highly conserved amino acid residues within FocA previously identified in structural studies are predicted to be important in the control of formate translocation. Here we present a first detailed in vivo analysis of these residues using a combined targeted amino acid exchange and formate-responsive lacZ fusion-based reporter approach. Sixteen exchanges were made and each variant was shown to be largely unaffected in its secondary and quaternary structure. The invariant H209 and T91 residues, which form part of the lower constriction site linking the Ω-loop with the pore cavity, proved to be important in governing the directionality of formate passage through the pore. A predicted salt-bridge triad of E208-K156-N213 along with the cytoplasmically-oriented N-terminal helix are also involved in pH-dependent gating of the channel. Together, our data are consistent with passive export and import of formate or formic acid through the channel.
authors.forename: Doreen
authors.initials: D
authors.lastname: Hunger
authors.forename: Claudia
authors.initials: C
authors.lastname: Doberenz
authors.forename: R Gary
authors.initials: RG
authors.lastname: Sawers
mesh_terms: D000595:Amino Acid Sequence; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; C088103:FocA protein, E coli; D005561:Formates; D026901:Membrane Transport Proteins; C030544:formic acid