FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity,High Affinity,Nucleoside and Nucleobase Transporter

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [³H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [³H]adenosine or [³H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.     

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp¹¹²) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.     

An early event in the transport mechanism of LacY protein: interaction between helices V and I

Helix V in LacY, which abuts and crosses helix I in the N-terminal helix bundle of LacY, contains Arg¹⁴⁴ and Trp¹⁵¹, two residues that play direct roles in sugar recognition and binding, as well as Cys¹⁵⁴, which is important for conformational flexibility. In this study, paired Cys replacement mutants in helices V and I were strategically constructed with tandem factor Xa protease cleavage sites in the loop between the two helices to test cross-linking. None of the mutants form disulfides spontaneously; however, three mutants (Pro²⁸ → Cys/Cys¹⁵⁴, Pro²⁸ → Cys/Val¹⁵⁸ → Cys, and Phe²⁹ → Cys/Val¹⁵⁸ → Cys) exhibit cross-linking after treatment with copper/1,10-phenanthroline (Cu/Ph) or 1,1-methanediyl bismethanethiosulfonate ((MTS)₂-1), 3–4 Å), and cross-linking is quantitative in the presence of ligand. Remarkably, with one mutant, complete cross-linking with (MTS)₂-1 has no effect on lactose transport, whereas quantitative disulfide cross-linking catalyzed by Cu/Ph markedly inhibits transport activity. The findings are consistant with a number of previous conclusions suggesting that sugar binding to LacY causes a localized scissors-like movement between helices V and I near the point where the two helices cross in the middle of the membrane. This ligand-induced movement may act to initiate the global conformational change resulting from sugar binding.     

Cytosolic Nucleotides Block and Regulate the Arabidopsis Vacuolar Anion Channel AtALMT9

The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al³⁺ to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.     

Path of nascent polypeptide in exit tunnel revealed by molecular dynamics simulation of ribosome

Molecular dynamics simulations were carried out on Thermus thermophilus 70S ribosome with and without a nascent polypeptide inside the exit tunnel. Modeling of the polypeptide in the tunnel revealed two possible paths: one over Arg⁹² of L22 and one under (from the viewpoint of 50S on top of 30S). A strong interaction between L4 and Arg⁹² was observed without the polypeptide and when it passed over Arg⁹². However, when the polypeptide passed under, Arg⁹² repositioned to interact with Ade²⁰⁵⁹ of 23S rRNA. Using steered molecular dynamics the polypeptide could be pulled through the L4-L22 constriction when situated under Arg⁹², but did not move when over. These results suggest that the tunnel is closed by the Arg⁹²-L4 interaction before elongation of the polypeptide and the tunnel leads the entering polypeptide from the peptidyl transferase center to the passage under Arg⁹², causing Arg⁹² to switch to an open position. It is possible, therefore, that Arg⁹² plays the role of a gate, opening and closing the tunnel at L4-L22. There is some disagreement over whether the tunnel is dynamic or rigid. At least within the timescale of our simulations conformational analysis showed that global motions mainly involve relative movement of the 50S and 30S subunits and seem not to affect the conformation of the tunnel.     

Effects of H-bonds on sugar binding to chitoporin from Vibrio harveyi

BackgroundVhChiP is a sugar-specific-porin present in the outer membrane of the marine bacterium Vibrio harveyi and responsible for chitin uptake, with a high selectivity for chitohexaose.MethodsVhChiP and its mutants were expressed and purified from BL21 (DE3) Omp8 Rosetta strain. After reconstitution into planar lipid bilayers, the ion current fluctuations caused by chitohexaose entering the channel were measured in deuterium oxide and in water.ResultsThe role of hydrogen-bonding in sugar binding was investigated by comparing channel occlusion by chitohexaose in buffers containing H₂O and D₂O. The BLM results revealed the significant contribution of hydrogen bonding to the binding of chitohexaose in the constriction zone of VhChiP. Replacing H₂O as solvent by D₂O significantly decreased the on- and off-rates of sugar penetration into the channel. The importance of hydrogen bonding inside the channel was more noticeable when the hydrophobicity of the constriction zone was diminished by replacing Trp¹³⁶ with the charged residues Asp or Arg. The on- and off-rates decreased up to 2.5-fold and 4-fold when Trp¹³⁶ was replaced by Arg, or 5-fold and 3-fold for Trp¹³⁶ replacement by Asp, respectively. Measuring the on-rate at different temperatures and for different channel mutants revealed the activation energy for chitohexaose entrance into VhChiP channel.ConclusionsHydrogen-bonds contribute to sugar permeation.     

Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using ⁶⁴Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher K_m value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and K_m values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu⁺ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.     

Functional Complementation and Genetic Deletion Studies of KirBac Channels: ACTIVATORY MUTATIONS HIGHLIGHT GATING-SENSITIVE DOMAINS*

The superfamily of prokaryotic inwardly rectifying (KirBac) potassium channels is homologous to mammalian Kir channels. However, relatively little is known about their regulation or about their physiological role in vivo. In this study, we have used random mutagenesis and genetic complementation in K⁺-auxotrophic Escherichia coli and Saccharomyces cerevisiae to identify activatory mutations in a range of different KirBac channels. We also show that the KirBac6.1 gene (slr5078) is necessary for normal growth of the cyanobacterium Synechocystis PCC6803. Functional analysis and molecular dynamics simulations of selected activatory mutations identified regions within the slide helix, transmembrane helices, and C terminus that function as important regulators of KirBac channel activity, as well as a region close to the selectivity filter of KirBac3.1 that may have an effect on gating. In particular, the mutations identified in TM2 favor a model of KirBac channel gating in which opening of the pore at the helix-bundle crossing plays a far more important role than has recently been proposed.     

Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1

Divalent metal-ion transporter-1 (DMT1) is a H⁺-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I_max/K_0.5 (determined from evoked currents at −70 mV): Cd²⁺ > Fe²⁺ > Co²⁺, Mn²⁺ ≫ Zn²⁺, Ni²⁺, VO²⁺. DMT1 expression did not stimulate the transport of Cr²⁺, Cr³⁺, Cu⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, or VO⁺. ⁵⁵Fe²⁺ transport was competitively inhibited by Co²⁺ and Mn²⁺. Zn²⁺ only weakly inhibited ⁵⁵Fe²⁺ transport. Our data reveal that DMT1 selects Fe²⁺ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.     

Functional Analysis of the Kv1.1 N255D Mutation Associated with Autosomal Dominant Hypomagnesemia

Mutations in the voltage-gated K⁺ channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936–942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, −41.5 ± 1.6, −45.5 ± 2.0, −50.5 ± 1.9, and −33.8 ± 1.3 mV to −29.4 ± 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 ± 0.2 ms) and N255V (5.2 ± 0.3 ms), and the hydrophilic mutant N255T (9.8 ± 0.4 ms) in comparison with wild type (13.0 ± 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted ∼13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.     

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca²⁺-ATPase. Upon binding to the Ca₂E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca²⁺ transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser¹⁸⁶ and Asp²⁰³ interact with Glu⁴³⁹ (N-domain) and Arg⁶⁷⁸ (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp²⁰³ and Arg⁶⁷⁸ rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser¹⁸⁶ and Glu⁴³⁹. By taking advantage of the ability of wild type and mutant Ca²⁺-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca²⁺-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp²⁰³-Arg⁶⁷⁸ and Ser¹⁸⁶-Glu⁴³⁹ interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser¹⁸⁶-Glu⁴³⁹ bond are mutually exclusive in the E2P ground state.     

Specificity of the Transport of Lipid II by FtsW in Escherichia coli

Synthesis of biogenic membranes requires transbilayer movement of lipid-linked sugar molecules. This biological process, which is fundamental in prokaryotic cells, remains as yet not clearly understood. In order to obtain insights into the molecular basis of its mode of action, we analyzed the structure-function relationship between Lipid II, the important building block of the bacterial cell wall, and its inner membrane-localized transporter FtsW. Here, we show that the predicted transmembrane helix 4 of Escherichia coli FtsW (this protein consists of 10 predicted transmembrane segments) is required for the transport activity of the protein. We have identified two charged residues (Arg¹⁴⁵ and Lys¹⁵³) within this segment that are specifically involved in the flipping of Lipid II. Mutating these two amino acids to uncharged ones affected the transport activity of FtsW. This was consistent with loss of in vivo activity of the mutants, as manifested by their inability to complement a temperature-sensitive strain of FtsW. The transport activity of FtsW could be inhibited with a Lipid II variant having an additional size of 420 Da. Reducing the size of this analog by about 274 Da resulted in the resumption of the transport activity of FtsW. This suggests that the integral membrane protein FtsW forms a size-restricted porelike structure, which accommodates Lipid II during transport across the bacterial cytoplasmic membrane.     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using β-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K_m for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.     

PH-induced collapse of the extracellular loops closes Escherichia coli maltoporin and allows the study of asymmetric sugar binding

LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli. Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH. Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4. When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis- or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pH- dependent closure of only one side of the channel. In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure. The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer. About 70-80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side). The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics. Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric. The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.     

Transmembrane Domain II of the Human Bile Acid Transporter SLC10A2 Coordinates Sodium Translocation

Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln⁷⁵, Phe⁷⁶, Met⁷⁹, Gly⁸³, Leu⁸⁶, Phe⁹⁰, and Asp⁹¹ in hASBT function. Computational analysis indicated that Asp⁹¹ may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr¹³⁴, Leu¹³⁸, and Thr¹⁴⁹) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln⁷⁵, Met⁷⁹, Thr⁸², and Leu⁸⁶ from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBT_NM), suggesting a universal mechanism for the SLC10A transporter family.     

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.     

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H⁺, like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H⁺-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.     

The Yeast Mitochondrial Citrate Transport Protein: MOLECULAR DETERMINANTS OF ITS SUBSTRATE SPECIFICITY*

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [¹⁴C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [¹⁴C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.