Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology.

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane-based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine KM, Vmax, KD, and kobs values for different sugar substrates. KD and kobs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H+ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H+ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s-1) and for sugar translocation (2 s-1 - 30 s-1 for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.

Mutation of two key aspartate residues alters stoichiometry of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae.

Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger's transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+ < 1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.

A channel profile report of the unusual K+ channel KtrB.

KtrAB is a key player in bacterial K+ uptake required for K+ homeostasis and osmoadaptation. The system is unique in structure and function. It consists of the K+-translocating channel subunit KtrB, which forms a dimer in the membrane, and the soluble regulatory subunit KtrA, which attaches to the cytoplasmic side of the dimer as an octameric ring conferring Na+ and ATP dependency to the system. Unlike most K+ channels, KtrB lacks the highly conserved T(X)GYG selectivity filter sequence. Instead, only a single glycine residue is found in each pore loop, which raises the question of how selective the ion channel is. Here, we characterized the KtrB subunit from the Gram-negative pathogen Vibrio alginolyticus by isothermal titration calorimetry, solid-supported membrane-based electrophysiology, whole-cell K+ uptake, and ACMA-based transport assays. We found that, despite its simple selectivity filter, KtrB selectively binds K+ with micromolar affinity. Rb+ and Cs+ bind with millimolar affinities. However, only K+ and the poorly binding Na+ are efficiently translocated, based on size exclusion by the gating loop. Importantly, the physiologically required K+ over Na+ selectivity is provided by the channel's high affinity for potassium, which interestingly results from the presence of the sodium ions themselves. In the presence of the KtrA subunit, sodium ions further decrease the Michaelis-Menten constant for K+ uptake from milli- to micromolar concentrations and increase the Vmax, suggesting that Na+ also facilitates channel gating. In conclusion, high binding affinity and facilitated K+ gating allow KtrAB to function as a selective K+ channel.

Coupling efficiency of secondary active transporters.

Secondary active transporters are fundamental to a myriad of biological processes. They use the electrochemical gradient of one solute to drive transport of another solute against its concentration gradient. Central to this mechanism is that the transport of one does not occur in the absence of the other. However, like in most of biology, imperfections in the coupling mechanism exist and we argue that these are innocuous and may even be beneficial for the cell. We discuss the energetics and kinetics of alternating-access in secondary transport and focus on the mechanistic aspects of imperfect coupling that give rise to leak pathways. Additionally, inspection of available transporter structures gives valuable insight into coupling mechanics, and we review literature where proteins have been altered to change their coupling efficiency.

Replacement of Lys-300 with a glutamine in the NhaA Na+/H+ antiporter of Escherichia coli yields a functional electrogenic transporter.

Much of the research on Na+/H+ exchange has been done in prokaryotic models, mainly on the NhaA Na+/H+-exchanger from Escherichia coli (EcNhaA). Two conserved aspartate residues, Asp-163 and Asp-164, are essential for transport and are candidates for possible binding sites for the two H+ that are exchanged for one Na+ to make the overall transport process electrogenic. More recently, a proposed mechanism of transport for EcNhaA has suggested direct binding of one of the transported H+ to the conserved Lys-300 residue, a salt bridge partner of Asp-163. This contention is supported by a study reporting that substitution of the equivalent residue, Lys-305, of a related Na+/H+ antiporter, NapA from Thermus thermophilus, renders the transporter electroneutral. In this work, we sought to establish whether the Lys-300 residue and its partner Asp-163 are essential for the electrogenicity of EcNhaA. To that end, we replaced Lys-300 with Gln, either alone or together with the simultaneous substitution of Asp-163 with Asn, and characterized these transporter variants in electrophysiological experiments combined with H+ transport measurements and stability analysis. We found that K300Q EcNhaA can still support electrogenic Na+/H+ antiport in EcNhaA, but has reduced thermal stability. A parallel electrophysiological investigation of the K305Q variant of TtNapA revealed that it is also electrogenic. Furthermore, replacement of both salt bridge partners in the ion-binding site of EcNhaA produced an electrogenic variant (D163N/K300Q). Our findings indicate that alternative mechanisms sustain EcNhaA activity in the absence of canonical ion-binding residues and that the conserved lysines confer structural stability.

A Loose Relationship: Incomplete H+/Sugar Coupling in the MFS Sugar Transporter GlcP.

The glucose transporter from Staphylococcus epidermidis, GlcPSe, is a homolog of the human GLUT sugar transporters of the major facilitator superfamily. Together with the xylose transporter from Escherichia coli, XylEEc, the other prominent prokaryotic GLUT homolog, GlcPSe, is equipped with a conserved proton-binding site arguing for an electrogenic transport mode. However, the electrophysiological analysis of GlcPSe presented here reveals important differences between the two GLUT homologs. GlcPSe, unlike XylEEc, does not perform steady-state electrogenic transport at symmetrical pH conditions. Furthermore, when a pH gradient is applied, partially uncoupled transport modes can be generated. In contrast to other bacterial sugar transporters analyzed so far, in GlcPSe sugar binding, translocation and release are also accomplished by the deprotonated transporter. Based on these experimental results, we conclude that coupling of sugar and H+ transport is incomplete in GlcPSe. To verify the viability of the observed partially coupled GlcPSe transport modes, we propose a universal eight-state kinetic model in which any degree of coupling is realized and H+/sugar symport represents only a specific instance. Furthermore, using sequence comparison with strictly coupled XylEEc and similar sugar transporters, we identify an additional charged residue that may be essential for effective H+/sugar symport.

SSM-Based Electrophysiology for Transporter Research.

Functional characterization of transport proteins using conventional electrophysiology can be challenging, especially for low turnover transporters or transporters from bacteria and intracellular compartments. Solid-supported membrane (SSM)-based electrophysiology is a sensitive and cell-free assay technique for the characterization of electrogenic membrane proteins. Purified proteins reconstituted into proteoliposomes or membrane vesicles from cell culture or native tissues are adsorbed to the sensor holding an SSM. A substrate or a ligand is applied via rapid solution exchange. The electrogenic transporter activity charges the sensor, which is recorded as a transient current. The high stability of the SSM allows cumulative measurements on the same sensor using different experimental conditions. This allows the determination of kinetic properties including EC50, IC50, Km, KD, and rate constants of electrogenic reactions. About 100 different transporters have been measured so far using this technique, among them symporters, exchangers, uniporters, ATP-, redox-, and light-driven ion pumps, as well as receptors and ion channels. Different instruments apply this technique: the laboratory setups use a closed flow-through arrangement, while the commercially available SURFE2R N1 resembles a pipetting robot. For drug screening purposes high-throughput systems, such as the SURFE2R 96SE enable the simultaneous measurement of up to 96 sensors.

Competition is the basis of the transport mechanism of the NhaB Na+/H+ exchanger from Klebsiella pneumoniae.

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.

From Gene to Function: Cell-Free Electrophysiological and Optical Analysis of Ion Pumps in Nanodiscs.

Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2's ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.

Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na+/H+ antiporter.

Na+/H+ antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na+, and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na+/H+ exchanger family and a model system for all related Na+/H+ exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H+ ions that NhaA exchanges for one Na+ ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length (i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na+ and H+ transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na+/H+ exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300.

Electrogenic Cation Binding in the Electroneutral Na+/H+ Antiporter of Pyrococcus abyssi.

Na+/H+ antiporters in the CPA1 branch of the cation proton antiporter family drive the electroneutral exchange of H+ against Na+ ions and ensure pH homeostasis in eukaryotic and prokaryotic organisms. Although their transport cycle is overall electroneutral, specific partial reactions are electrogenic. Here, we present an electrophysiological study of the PaNhaP Na+/H+ antiporter from Pyrococcus abyssi reconstituted into liposomes. Positive transient currents were recorded upon addition of Na+ to PaNhaP proteoliposomes, indicating a reaction where positive charge is rapidly displaced into the proteoliposomes with a rate constant of k >200 s-1 We attribute the recorded currents to an electrogenic reaction that includes Na+ binding and possibly occlusion. Subsequently, positive charge is transported out of the cell associated with H+ binding, so that the overall reaction is electroneutral. We show that the differences in pH profile and Na+ affinity of PaNhaP and the related MjNhaP1 from Methanocaldococcus jannaschii can be attributed to an additional negatively charged glutamate residue in PaNhaP. The results are discussed in the context of the physiological function of PaNhaP and other microbial Na+/H+ exchangers. We propose that both, electroneutral and electrogenic Na+/H+ antiporters, represent a carefully tuned self-regulatory system, which drives the cytoplasmic pH back to neutral after any deviation.

pH Regulation of Electrogenic Sugar/H+ Symport in MFS Sugar Permeases.

Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6-7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.

Functional characterization of solute carrier (SLC) 26/sulfate permease (SulP) proteins in membrane mimetic systems.

Solute carrier (SLC) 26 or sulfate permease (SulP) anion transporters, belong to a phylogenetically ancient family of secondary active transporters. Members of the family are involved in several human genetic diseases and cell physiological processes. Despite their importance, the substrates for transport by this family of proteins have been poorly characterized. In this study, recombinant StmYchM/DauA, a SulP from Salmonella typhimurium was purified to homogeneity and functionally characterized. StmYchM/DauA was found to be a dimer in solution as determined by size exclusion chromatography coupled to multiple angle light scattering. We report a functional characterization of the SulP proteins in two membrane mimetic systems and reveal a dual nature of anionic substrates for SulP. StmYchM/DauA functionally incorporated into nanodiscs could bind fumarate with millimolar affinities (KD = 4.6 ± 0.29 mM) as detected by intrinsic tryptophan fluorescence quench studies. In contrast, electrophysiological experiments performed in reconstituted liposomes indicate a strong bicarbonate transport in the presence of chloride but no detectable electrogenic fumarate transport. We hence suggest that while SulP acts as an electrogenic bicarbonate transporter, fumarate may serve as substrate under different conditions indicating multiple functions of SulP.

Structural and Functional Studies of NirC from Salmonella typhimurium.

NirC is a pentameric transport system for monovalent anions that is expressed in the context of assimilatory nitrite reductase NirBD in a wide variety of enterobacterial species. A NirC pentamer contains individual pores in each protomer that mediate the passage of at least the nitrite [Formula: see text] and nitrate [Formula: see text] anions. As a member of the formate/nitrite transporter family of membrane transport proteins, NirC shares a range of structural and functional features with the formate channel FocA and the hydrosulfide channel AsrD (HSC). NirC from the enteropathogen Salmonella typhimurium has been studied by X-ray crystallography, proton uptake assays, and different electrophysiological techniques, and the picture that has emerged shows a fast and versatile transport system for nitrite that doubles as a defense system during the enteric life of the bacterium. Structural and functional assays are described, which shed light on the transport mechanism of this important molecular machine.

A universal mechanism for transport and regulation of CPA sodium proton exchangers.

Recent studies performed on a series of Na+/H+ exchangers have led us to postulate a general mechanism for Na+/H+ exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na+/H+ exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na+/H+ exchangers.

Species differences in bacterial NhaA Na+/H+ exchangers.

Bacteria have adapted their NhaA Na(+)/H(+) exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H(+) and Na(+). We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.

Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium.

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

NhaA Na+/H+ antiporter mutants that hardly react to the membrane potential.

pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70-95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.

Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers.

Na(+)/H(+) exchangers are essential for regulation of intracellular proton and sodium concentrations in all living organisms. We examined and experimentally verified a kinetic model for Na(+)/H(+) exchangers, where a single binding site is alternatively occupied by Na(+) or one or two H(+) ions. The proposed transport mechanism inherently down-regulates Na(+)/H(+) exchangers at extreme pH, preventing excessive cytoplasmic acidification or alkalinization. As an experimental test system we present the first electrophysiological investigation of an electroneutral Na(+)/H(+) exchanger, NhaP1 from Methanocaldococcus jannaschii (MjNhaP1), a close homologue of the medically important eukaryotic NHE Na(+)/H(+) exchangers. The kinetic model describes the experimentally observed substrate dependences of MjNhaP1, and the transport mechanism explains alkaline down-regulation of MjNhaP1. Because this model also accounts for acidic down-regulation of the electrogenic NhaA Na(+)/H(+) exchanger from Escherichia coli (EcNhaA, shown in a previous publication) we conclude that it applies generally to all Na(+)/H(+) exchangers, electrogenic as well as electroneutral, and elegantly explains their pH regulation. Furthermore, the electrophysiological analysis allows insight into the electrostatic structure of the translocation complex in electroneutral and electrogenic Na(+)/H(+) exchangers.

Electrophysiological characterization of uncoupled mutants of LacY.

In this study of the lactose permease of Escherichia coli (LacY), five functionally irreplaceable residues involved specifically in H(+) translocation (Arg302 and Glu325) or in the coupling between protonation and sugar binding (Tyr236, Glu269, and His322) were mutated individually or together with mutant Glu325 → Ala. The wild type and each mutant were purified and reconstituted into proteoliposomes, which were then examined using solid-supported-membrane-based electrophysiology. Mutants Glu325 → Ala or Arg302 → Ala, in which H(+) symport is abolished, exhibit a weakly electrogenic rapid reaction triggered by sugar binding. The reaction is essentially absent in mutant Tyr236 → Phe, Glu269 → Ala, and His322 → Ala, and each of these mutations blocks the electrogenic reaction observed in the Glu325 → Ala mutant. The findings are consistent with the interpretation that the electrogenic reaction induced by sugar binding is due to rearrangement of charged residues in LacY and that this reaction is blocked by mutation of each member of the Tyr236/Glu269/His322 triad. In addition, further support is provided for the conclusion that deprotonation is rate limiting for downhill lactose/H(+) symport.