Single-channel measurements of an N-acetylneuraminic acid-inducible outer membrane channel in Escherichia coli.

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.

NanC crystal structure, a model for outer-membrane channels of the acidic sugar-specific KdgM porin family.

Sialic acids are acidic sugars present mostly on vertebrate cell surfaces, which can be metabolized by bacteria and act as an inflammation signal. N-Acetylneuraminic acid, the most abundant sialic acid, can enter into Escherichia coli K12 through NanC, an N-acetylneuraminic acid-inducible outer-membrane channel. With its 215 residues, NanC belongs to the family of small monomeric KdgM-related porins. KdgM homologues are found in gammaproteobacteria, including major plant and human pathogens, and together they define a large family of putative acidic sugar/oligosaccharide transporters, which are as yet poorly characterized. Here, we present the first high-resolution structure of a KdgM family member. NanC folds into a 28-A-high, 12-stranded beta-barrel, resembling the beta-domain of autotransporter NalP and defining an open pore with an average radius of 3.3 A. The channel is lined by two strings of basic residues facing each other across the pore, a feature that appears largely conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.