Characterization of the nucleoside-binding site inside the Tsx channel of Escherichia coli outer membrane. Reconstitution experiments with lipid bilayer membranes.
Reconstitution of purified Tsx protein from Escherichia coli into lipid bilayer membranes showed that Tsx formed small ion-permeable channels with a single-channel conductance of 10 pS in 1 M KCl. The dependence of conductance versus salt concentration was linear, suggesting that Tsx has no binding site for ions. Conductance was inhibited by the addition of 20 mM adenosine. Titration of the Tsx-mediated membrane conductance with different solutes including free bases, nucleosides, and deoxynucleosides suggested that the channel contains a binding site for nucleosides but not for sugars or amino acids, and binding increased in the following order: free base, nucleoside, and deoxynucleoside. Among the five nucleosides the stability constant for the binding increased in the order of cytidine, guanosine, uridine, adenosine, and thymidine. Control experiments revealed that the binding of the nucleosides is independent of ion concentration in the aqueous phase, i.e. there was no competition between nucleosides and ions for the binding site inside the channel. The binding of the solutes to the channel interior can be explained by a one-site two-barrier model for the Tsx channel. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion pore.
Benz
R
R
Lehrstuhl für Biotechnologie, Universität Würzburg, Federal Republic of Germany.
Schmid
A
A
Maier
C
C
Bremer
E
E
D000241:Adenosine / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D002463:Cell Membrane Permeability; D004553:Electric Conductivity / Q000187:drug effects; D004926:Escherichia coli / Q000032:analysis*; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008564:Membrane Potentials; D009705:Nucleosides / Q000378:metabolism*; D012492:Salts / Q000378:metabolism
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008563:Membrane Lipids; D009705:Nucleosides; D012492:Salts; D000241:Adenosine