Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes.

Delineating electrogenic reactions during lactose/H+ symport.

Electrogenic reactions accompanying downhill lactose/H(+) symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.

Electrophysiological characterization of LacY.

Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H(+) symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY.

SSM-based electrophysiology.

An assay technique for the electrical characterization of electrogenic transport proteins on solid supported membranes is presented. Membrane vesicles, proteoliposomes or membrane fragments containing the transporter are adsorbed to the solid supported membrane and are activated by providing a substrate or a ligand via a rapid solution exchange. This technique opens up new possibilities where conventional electrophysiology fails like transporters or ion channels from bacteria and from intracellular compartments.

Rapid activation of the melibiose permease MelB immobilized on a solid-supported membrane.

Rapid solution exchange on a solid-supported membrane (SSM) is investigated using fluidic structures and a solid-supported membrane of 1 mm diameter in wall jet geometry. The flow is analyzed with a new technique based on specific ion interactions with the surface combined with an electrical measurement. The critical parameters affecting the time course of the solution exchange and the transfer function describing the time resolution of the SSM system are determined.

Specific anion and cation binding to lipid membranes investigated on a solid supported membrane.

Ion binding to a lipid membrane is studied by application of a rapid solution exchange on a solid supported membrane. The resulting charge displacement is analyzed in terms of the affinity of the applied ions to the lipid surface. We find that chaotropic anions and kosmotropic cations are attracted to the membrane independent of the membrane composition.

Electrostatic properties and macroscopic electrodiffusion in OmpF porin and mutants.

The bacterial porin OmpF found in the outer membrane of E. coli is a wide channel, characterized by its poor selectivity and almost no ion specificity. It has an asymmetric structure, with relatively large entrances and a narrow constriction.