Shimon Schuldiner

A cDNA that suppresses MPP+ toxicity encodes a vesicular amine transporter

Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinsons disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.

Mechanism of Transport and Storage of Neurotransmitter

This review will focus on the bioenergetics, mechanism, and molecular basis of neurotransmitter transport. As indicated in the next section, these processes play an important role in the overall process of synaptic transmission. During the last few years, direct evidence has been obtained that these processes are coupled chemiosmotically, i.e., the accumulation of neurotransmitters is driven by ion gradients. Two types of neurotransmitter transport systems have been identified: sodium-coupled systems located in the synaptic plasma membrane of nerves (and sometimes in the plasma membrane of glial cells) and proton-coupled systems which are part of the membrane of intracellular storage organelles. From a bioenergetic point of view, the sodium-coupled systems are especially interesting, since it has recently been discovered that many systems require other ions in addition to sodium. It has now been demonstrated in several cases that, besides sodium ions, these additional ions, such as chloride and potassium, serve as additional coupling ions. These systems will be reviewed here in considerable detail with emphasis on the role of the additional ions. In the second part of the review we shall focus on neurotransmitter transport into storage organelles. Although both sodium and proton coupled systems have been reviewed in the past, there has been a shift from a kinetic and thermodynamic to a biochemical approach. In fact, a few transporters have been identified and functionally reconstituted. These developments have of course been incorporated in this review.

Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer.

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton‐linked efflux. We have determined the three‐dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 Å resolution. The structure of EmrE consists of a bundle of eight transmembrane α‐helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent‐solubilized protein binds tetraphenylphosphonium (TPP+) with a KD of 10 nM. One mole of ligand is bound per ∼3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of ∼7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane‐embedded charged residue shows little transport activity, but binds TPP+ at levels similar to those of the wild‐type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.

A Molecular Glimpse of Vesicular Monoamine Transporters

Synaptic transmission involves the regulated release of transmitter molecules to the synaptic cleft, where they interact with postsynaptic receptors that subsequently transduce the information. Removal of the transmitter from the cleft enables termination of the signal, and it usually occurs by its reuptake back to the presynaptic terminal or into glial elements in a sodium-dependent process. This process assures constant and high levels of neurotransmitters in the neuron and low concentrations in the cleft. Neurotransmitters are stored in subcellular organelles so as to ensure their regulated release. This storage process also protects the accumulated molecules from leakage or intraneuronal metabolism, and it also protects the neuron from possible toxic effects of the transmitters. Also, the removal of intraneuronal molecules into the storage system effectively lowers the concentration gradient across the neuronal membrane and thus acts as an amplification stage for the overall process of uptake. Drugs that interact with either transport system have profound pharmacological effects as they modify the levels of neurotransmitter in the cleft. Inhibitors that interfere with these activities include the tricyclic antidepressants, stimulants such as amphetamines and cocaine, antihypertensives such as reserpine, and neurotoxins such as N-methyl-4-phenylpyridinium (MPP+). Plasma membrane transporters have been intensively studied at the mechanistic, biochemical, and molecular levels. The molecular characterization began with the purification, amino acid sequencing, and cloning of the y-aminobutyric acid (GABA) transporter (Guastella et al., 1990), and since it has become clear that these Na+and C1--coupled transporters represent a group of integral membrane proteins encoded by a closely related family of genes that includes the transporters for biogenic amines, GABA, glycine, proline, choline, and taurine (Amara and Kuhar, 1993; Rudnick and Clark, 1993). A different and novel class of plasma membrane transporters is represented by the glutamate transporter (Pines et al., 1992; Storck et al., 1992; Kanner, 1993). Vesicular transport has been observed for several classical transmitters, including acetylcholine (Toll and Howard, 1980; Anderson et al., 1982), the amines (Njus et al., 1986; Johnson, 1988), glutamate (Shioi et al., 1989; Maycox et al., 1990; Tabb et al., 1992), and GABA and glycine (Kish et al., 1989; Burger et al., 1991; Thomasreetz et al., 1993). The amine transporter has been the one most intensively studied and is the one for which most molecular information has been obtained (Njus et al., 1986; Kanner and Schuldiner, 1987; Johnson, 1988). The key for this knowledge resides in the availability of an excellent experimental paradigm for the study of this system, the chromaffin granules of the adrenal medulla, and potent and specific inhibitors, such as reserpine and tetrabenazine (TBZ). This review will stress some of the original information from work performed with the native chromaffin granule membrane and the purified transporter from bovine adrenal. Selected aspects related to the recent cloning of cDNAs coding for various monoamine transporters (Erickson et al., 1992; Liu et al., 1992; Surratt et al., 1993) and a putative acetycholine transporter (Alfonso et al., 1993) will be emphasized. Finally, the evolving concept that monoamine transporters may function as multidrug transporters will be discussed.

The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution.

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo‐electron microscopy and a projection structure of EmrE was calculated to 7 Å resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions ∼31 × 40 Å, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2‐fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.

A coordinated network of transporters with overlapping specificities provides a robust survival strategy

Multidrug transporters provide a survival strategy for living organisms. As expected given their central role in survival, these transporters are ubiquitous, and in many genomes, several genes coding for putative transporters have been identified. However, in an organism such as Escherichia coli mutations in genes coding for transporters other than the major AcrAB-TolC multidrug efflux transporter have only a marginal effect on phenotype. Thus, whether the physiological role of the transporters identified is indeed drug export has been questioned. We show here that the minor effect of single mutations is due to the overlapping functionality of several transporters. This was revealed by generating multiple chromosomal deletion mutations in genes coding for transporters that share the same substrate and testing their effect on the resistance phenotype. In addition, complementation studies imply that AcrAB-TolC confers robust resistance provided that single-component transporters in the plasma membrane are functional. This finding supports the contention that hydrophobic drugs are removed in a 2-stage process: AcrAB-TolC removes substrates from the periplasmic space, while single-component transporters remove them from the cell. The overlapping specificities of the transporters ensure coverage of a wide range of xenobiotics and provide robustness in the response to environmental stress. This strategy also confers evolvability to the organism by reducing constraints on change and allowing the accumulation of nonlethal variation.

EmrE, a model for studying evolution and mechanism of ion-coupled transporters.

EmrE is a small (110 residues) SMR transporter from Escherichia coli that extrudes positively charged aromatic drugs in exchange for two protons, thus rendering bacteria resistant to a variety of toxic compounds. Due to its size, stability and retention of its function upon solubilization in detergent, EmrE provides a unique experimental paradigm for the biochemical and biophysical studies of membrane based ion-coupled transporters. In addition, EmrE has been in center stage in the past two years because it provides also a paradigm for the study of the evolution of membrane proteins. Controversy around this topic is still going on and some novel concepts are surfacing that may contribute to our understanding of evolution of topology of membrane proteins. Furthermore, based on the findings that the cell multidrug transporters interact functionally we introduce the concept of a cell Resistosome.

Negative Dominance Studies Demonstrate the Oligomeric Structure of EmrE, a Multidrug Antiporter from Escherichia coli

EmrE, the smallest known ion-coupled transporter, is an Escherichia coli 12-kDa protein 80% helical and soluble in organic solvents. EmrE is a polyspecific antiporter that exchanges hydrogen ions with aromatic toxic cations such as methyl viologen. Since it is many times smaller than the classical consensus 12 transmembrane segments transporters, it was particularly interesting to determine its oligomeric state. For this purpose, a series of nonfunctional mutants has been generated and characterized to test their effect on the activity of the wild-type protein upon mixing. As opposed to the wild type, these mutants do not confer resistance to methyl viologen, ethidium bromide, or a series of other toxicants. Co-expression of each of the nonfunctional mutants with the wild-type protein results in a reduction in the ability of the functional transporter to confer resistance to several toxicants. To perform mixing experiments in vitro, all the mutants have been purified by extraction with organic solvents, reconstituted in proteoliposomes, and found to be inactive. When co-reconstituted with wild-type protein, they inhibit the activity of the latter in a dose-dependent form up to full inhibition. We assume that this inhibition is due to the formation of mixed oligomers in which the presence of one nonfunctional subunit causes full inactivation. A binomial analysis of the results based on the latter assumptions do not provide statistically significant answers but suggests that the oligomer is composed of three subunits. The results described provide the first in vitro demonstration of the functional oligomeric structure of an ion-coupled transporter.

Projection structure of NhaA, a secondary transporter from Escherichia coli, at 4.0 A resolution.

Electron cryomicroscopy of frozen‐hydrated two‐dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane α‐helices, has facilitated the calculation of a projection map of NhaA at 4.0 Å resolution. NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two‐dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of ∼38×48 Å with a ring‐shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.