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. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

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. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H(+) symport is H(+) release. In contrast, LacY mutants Glu-325-->Ala and Cys-154-->Gly, which bind ligand normally, but are severely defective with respect to lactose/H(+) symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H(+) symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H(+) during downhill sugar/H(+) symport, which is the limiting step for this mode of transport.

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 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H(+) translocation is abolished either by acidic pH or by a Glu325 --> Ala mutation in the H(+) binding site (this step with a rate constant of approximately 200 s(-1) for wild-type LacY leads to an intermediate proposed to represent an "occluded" state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of approximately 30 s(-1) and a pK of 7.5. This partial reaction is assigned to rate-limiting H(+) release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical-biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.

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. Its rugged design and potential for automation make it suitable for drug screening.

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. By applying continuum electrostatic methods we determine the ionization states of titratable amino acid residues in the protein and calculate self-consistently the electric potential 3-D distribution within the channel. The average electrostatic properties are then represented by an effective fixed charge distribution along the pore which is the input for a macroscopic electrodiffusion model. The theoretical predictions agree with measurements performed under different salt gradients and pH. The sensitivity of reversal potential and conductance to the direction of the salt gradient and the solution pH is captured by the model. The theory is also able to explain the influence of the lipid membrane charge. The same methodology is satisfactorily applied to some OmpF mutants involving slight structural changes but a large number of net charges. The correlation found between atomic structure and ionic selectivity shows that the transport characteristics of wide channels like OmpF and its mutants are mainly regulated by the collective action of a large number of residues, rather than by the specific interactions of residues at particular locations.

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. The experimental data indicate that solution transport represents an intermediate situation between the plug flow and the Hagen-Poiseuille laminar flow regime. However, to a good approximation the rise of the surface concentration can be described by Hagen-Poiseuille flow with ideal mixing at the surface of the SSM. Using an improved cuvette design, solution exchange as fast as 2 ms was achieved at the surface of a solid-supported membrane. As an application of the technique, the rate constant of a fast electrogenic reaction in the melibiose permease MelB, a bacterial ( Escherichia coli) sugar transporter, is determined. For comparison, the kinetics of a conformational transition of the same transporter was measured using stopped-flow tryptophan fluorescence spectroscopy. The relaxation time constant obtained for the charge displacement agrees with that determined in the stopped-flow experiments. This demonstrates that upon sugar binding MelB undergoes an electrogenic conformational transition with a rate constant of k approximately 250 s (-1).

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. In particular, the same behavior is found for lipid headgroups bearing no charge, like monoolein. This general trend is modulated by electrostatic interaction of the ions with the lipid headgroup charge. These results cannot be explained with the current models of specific ion interactions.