title: Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli.
abstract: NanC is an Escherichia coli outer membrane protein involved in sialic acid (Neu5Ac, i.e., N-acetylneuraminic acid) uptake. Expression of the NanC gene is induced and controlled by Neu5Ac. The transport mechanism of Neu5Ac is not known. The structure of NanC was recently solved (PDB code: 2WJQ) and includes a unique arrangement of positively charged (basic) side chains consistent with a role in acidic sugar transport. However, initial functional measurements of NanC failed to find its role in the transport of sialic acids, perhaps because of the ionic conditions used in the experiments. We show here that the ionic conditions generally preferred for measuring the function of outer-membrane porins are not appropriate for NanC. Single channels of NanC at pH 7.0 have: (1) conductance 100 pS to 800 pS in 100 mM: KCl to 3 M: KCl), (2) anion over cation selectivity (V (reversal) = +16 mV in 250 mM: KCl || 1 M: KCl), and (3) two forms of voltage-dependent gating (channel closures above ± 200 mV). Single-channel conductance decreases by 50% when HEPES concentration is increased from 100 μM: to 100 mM: in 250 mM: KCl at pH 7.4, consistent with the two HEPES binding sites observed in the crystal structure. Studying alternative buffers, we find that phosphate interferes with the channel conductance. Single-channel conductance decreases by 19% when phosphate concentration is increased from 0 mM: to 5 mM: in 250 mM: KCl at pH 8.0. Surprisingly, TRIS in the baths reacts with Ag|AgCl electrodes, producing artifacts even when the electrodes are on the far side of agar-KCl bridges. A suitable baseline solution for NanC is 250 mM: KCl adjusted to pH 7.0 without buffer.
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University, 1750 W. Harrison St., Chicago, IL 60612, USA.
authors.forename: Janhavi
authors.initials: J
authors.lastname: Giri
authors.forename: John M
authors.initials: JM
authors.lastname: Tang
authors.forename: Christophe
authors.initials: C
authors.lastname: Wirth
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Peneff
authors.forename: Bob
authors.initials: B
authors.lastname: Eisenberg
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001692:Biological Transport; D002021:Buffers; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism / Q000494:pharmacology*; D018272:Porins / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002021:Buffers; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; C528455:NanC protein, E coli; D018272:Porins; D011189:Potassium Chloride; D019158:N-Acetylneuraminic Acid