Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters

Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.     

Characterization of a functional bacterial homologue of sodium-dependent neurotransmitter transporters

The tnaT gene of Symbiobacterium thermophilum encodes a protein homologous to sodium-dependent neurotransmitter transporters. Expression of the tnaT gene product in Escherichia coli conferred the ability to accumulate tryptophan from the medium and the ability to grow on tryptophan as a sole source of carbon. Transport was Na(+)-dependent and highly selective. The K(m) for tryptophan was approximately 145 nm, and tryptophan transport was unchanged in the presence of 100 microM concentrations of other amino acids. Tryptamine and serotonin were weak inhibitors with K(I) values of 200 and 440 microM, respectively. By using a T7 promoter-based system, TnaT with an N-terminal His(6) tag was expressed at high levels in the membrane and was purified to near-homogeneity in high yield.     

Predicting functional sites in proteins: site-specific evolutionary models and their application to neurotransmitter transporters

Currently there exist several computational methods for predicting the functional sites in a set of homologous proteins based on their sequences. Due to difficulties in defining the functional site in a protein, it is not trivial to compare the performance of these methods, evaluate their limitations and quantify improvements by new approaches. Here, we use extensive mutation data from two proteins, Lac repressor and subtilisin, to perform such an analysis. Along with the evaluation of existing approaches, we describe a site class model of evolution as a tool to predict functional sites in proteins. The results indicate that this model, which simulates the evolution process at the amino acid level using site-specific substitution matrices, provides the most accurate information on functional sites in a given protein family. Secondly, we present an application of this model to neurotransmitter transporters, a superfamily of proteins of which we have limited experimental knowledge. Based on this application we present testable hypotheses regarding the mechanism of action of these proteins.     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system(CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid reuptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter     

Glycine neurotransmitter transporters: an update

Glycine accomplishes several functions as a transmitter in the central nervous system (CNS). As an inhibitory neurotransmitter, it participates in the processing of motor and sensory information that permits movement, vision, and audition. This action of glycine is mediated by the strychnine-sensitive glycine receptor, whose activation produces inhibitory post-synaptic potentials. In some areas of the CNS, glycine seems to be co-released with GABA, the main inhibitory amino acid neurotransmitter. In addition, glycine modulates excitatory neurotransmission by potentiating the action of glutamate at N-methyl-D-aspartate (NMDA) receptors. It is believed that the termination of the different synaptic actions of glycine is produced by rapid re-uptake through two sodium-and-chloride-coupled transporters, GLYT1 and GLYT2, located in the plasma membrane of glial cells or pre-synaptic terminals, respectively. Glycine transporters may become major targets for therapeutic of pathological alterations in synaptic function. This article reviews recent progress on the study of the molecular heterogeneity, localization, function, structure, regulation and pharmacology of the glycine transporter proteins.     

ABC transporters: bacterial exporters.

The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review.     

Single-file diffusion and neurotransmitter transporters: Hodgkin and Keynes model revisited

Norepinephrine transporters (NETs) use the Na gradient to remove norepinephrine (NE) from the synaptic cleft of adrenergic neurons following NE release from the presynaptic terminal. By coupling NE to the inwardly directed Na gradient, it is possible to concentrate NE inside cells. This mechanism, which is referred to as co-transport or secondary transport (L?uger, 1991, Electrogenic Ion Pumps, Sinauer Associates) is apparently universal: Na coupled transport applies to serotonin transporters (SERTs), dopamine transporters (DATs), glutamate transporters, and many others, including transporters for osmolites, metabolites and substrates such as sugar. Recently we have shown that NETs and SERTs transport norepinephrine or serotonin as if Na and the transmitter permeated through an ion channel together 'Galli et al., 1998, PNAS 95, 13260-13265; Petersen and DeFelice, 1999, Nature Neurosci. 2, 605-610'. These data are paradoxical because it has been difficult to envisage how NE, for example, would couple to Na if these ions move passively through an open pore. An 'alternating access' model is usually evoked to explain coupling: in such models NE and Na bind to NET, which then undergoes a conformational change to release NE and Na on the inside. The empty transporter then turns outward to complete the cycle. Alternating-access models never afford access to an open channel. Rather, substrates and co-transported ions are occluded in the transporter and carried across the membrane. The coupling mechanism we propose is fundamentally different than the coupling mechanism evoked in the alternating access model. To explain coupling in co-transporters, we use a mechanism first evoked by 'Hodgkin and Keynes (1955) J. Physiol. 128, 61-88' to explain ion interactions in K-selective channels. In the Hodgkin and Keynes model, K ions move single-file through a long narrow pore. Their model accounted for the inward/outward flux ratio if they assumed that two K ions queue within the pore. We evoke a similar model for the co-transport of transmitter and Na. In our case, however, coupling occurs not only between like ions but also between unlike ions (i.e. the transmitter and Na ). We made a replica of the Hodgkin and Keynes mechanical model to test our ideas, and we extended the model with computer simulations using Monte Carlo methods. We also developed an analytic formula for Na coupled co-transport that is analogous to the single-file Ussing equation for channels. The model shows that stochastic diffusion through a long narrow pore can explain coupled transport. The length of the pore amplifies the Na gradient that drives co-transport.     

Amino acid neurotransmitter transporters: Structure,function, and molecular diversity

Many biologically active compounds including neurotransmitters, metabolic precursors, and certain drugs are accumulated intracellularly by transporters that are coupled to the transmembrane Na⁺ gradient. Amino acid neurotransmitter transporters play a key role in the regulation of extracellular amino acid concentrations and termination of neurotransmission in the CNS

1 Abbreviations: CNS, central nervous system; GABA, γ-aminobutyric acid; cDNA, complementary deoxyribonucleic acid; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; DAG, diacyl glycerol; R59022, DAG kinase inhibitor; AA, arachidonic acid; ACHC, cis-3-aminocyclohexanecarboxylic acid; GAT-A, ACHC-sensitive GABA transporter; GAT-B, β-alanine-sensitive GABA transporter; GLY-1 and GLYT-1, glycine transporters; PROT-1, proline transporter; BGT-1, betaine transporter.

. Transporters for the major amino acid neurotransmitters glutamate, GABA, and glycine are found in both neurons and glial cells. Recent work has resulted in the identification of cDNAs encoding several amino acid neurotransmitter transport proteins, all of which belong to the Na⁺-and Cl^?-dependent transporter gene family. The diversity of this family suggests a degree of transporter heterogeneity that is greater than that indicated by biochemical and pharmacological studies.     

ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters.

The traffic ATPases superfamily includes known transporters, both prokaryotic and eukaryotic, including the medically important proteins, P-glycoprotein, and the cystic fibrosis gene product (CFTR), which is known to be a Cl- channel. The structure and mechanism of action of the best-studied members of the superfamily, the periplasmic permeases, are described and related to that of CFTR and eukaryotic traffic ATPases in general. The contention is put forward that the distinction between the architecture and mechanisms of action of channels and transporters is blurred.     

Colocalization of different neurotransmitter transporters on synaptic vesicles is sparse except for VGLUT1 and ZnT3

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Sodium-dependent neurotransmitter transporters: oligomerization as a determinant of transporter function and trafficking

Constitutive oligomer formation appears to be the rule for the neurotransmitter:sodium symporter (NSS) family of proteins. The propensity to form oligomers is a prerequisite for NSS proteins to pass the rigid mechanisms of quality control in the endoplasmic reticulum. Moreover, recent findings suggest that correct trafficking to the plasma membrane appears to rely on the interaction of NSS homo-oligomers with components of the COPII-vesicle machinery. The transporters present at the plasma membrane are most likely organized in a tetrameric arrangement, as a dimer of dimers. In this review, we will address ongoing efforts to unravel the underlying mechanisms of oligomer formation at the molecular and cellular levels, and we will discuss oligomerization in terms of transporter function.     

Dynamics of surface neurotransmitter receptors and transporters in glial cells: Single molecule insights

The surface dynamics of neurotransmitter receptors and transporters, as well as ion channels, has been well-documented in neurons, revealing complex molecular behaviour and key physiological functions. However, our understanding of the membrane trafficking and dynamics of the signalling molecules located at the plasma membrane of glial cells is still in its infancy. Yet, recent breakthroughs in the field of glial cells have been obtained using combination of superresolution microscopy, single molecule imaging, and electrophysiological recordings. Here, we review our current knowledge on the surface dynamics of neurotransmitter receptors, transporters and ion channels, in glial cells. It has emerged that the brain cell network activity, synaptic activity, and calcium signalling, regulate the surface distribution and dynamics of these molecules. Remarkably, the dynamics of a given neurotransmitter receptor/transporter at the plasma membrane of a glial cell or neuron is unique, revealing the existence of cell-type specific regulatory pathways. Thus, investigating the dynamics of signalling proteins at the surface of glial cells will likely shed new light on our understanding of glial cell physiology and pathology.     

Neuronal development: neighbors have to be different

The assembly of neurons into functional circuits requires a multitude of cellular recognition events. Recent work on the hypervariable Drosophila Dscam gene revealed how a vast number of cell adhesion proteins contributes to neuronal patterning.     

Mystery of multidrug transporters: the answer can be simple

Multidrug efflux transporters, found in all living cells and protecting them from multiple structurally dissimilar hydrophobic toxins, have fascinated researchers for decades and presented a number of puzzling questions. These transporters demonstrate a remarkably broad substrate specificity, which seemingly contradicts established dogmas of biochemistry. Although sharing highly unusual properties, in some unexplained way, they have arisen multiple times in the evolution of several families of membrane proteins. Furthermore, the number of multidrug transporters encoded in each genome is so large that their role in cellular physiology has remained un-certain. Recent advances in the structural analysis of a number of soluble multidrug-recognizing proteins show that these proteins possess large hydrophobic binding sites and bind their substrates through a combination of a hydrophobic effect and electrostatic attraction, rather than by establishing a precise network of hydrogen bonds and other specific interactions characteristic of traditionally studied enzymes and receptors. Low-resolution structural studies of multidrug transporters suggest that they possess similar large binding sites and may use similar simple principles of substrate recognition. This would explain not only their broad substrate specificity, but also their unusual evolutionary relationships and the apparent multiplicity in genomes of organisms of all evolutionary kingdoms. Although further structural studies will be needed to prove this hypothesis, it is already clear that the explanation of the puzzling phenomenon of multidrug efflux may not necessarily require any substantially new biochemical or biological principles.     

Hard ticks and their bacterial endosymbionts (or would be pathogens)

The symbiotic microorganisms of arthropod vectors are highly significant from several points of view, partly due to their possible roles in the transmission of pathogenic causative agents by blood-sucking vectors. Although ticks are well studied because of their significance to human health, novel microbial associations remain to be described. This review summarises several endosymbiotic bacterial species in hard ticks from various parts of the world, including Coxiella-, Francisella-, Rickettsia- and Arsenophonus-like symbionts as well as Candidatus Midichloria mitochondrii and Wolbachia. New methodologies for the isolation and characterization of tick-associated bacteria will, in turn, encourage new strategies of tick control by studying their endosymbionts.     

Making ends meet: repairing breaks in bacterial DNA by non-homologous end-joining

DNA double-strand breaks (DSBs) are one of the most dangerous forms of DNA lesion that can result in genomic instability and cell death. Therefore cells have developed elaborate DSB-repair pathways to maintain the integrity of genomic DNA. There are two major pathways for the repair of DSBs in eukaryotes: homologous recombination and non-homologous end-joining (NHEJ). Until very recently, the NHEJ pathway had been thought to be restricted to the eukarya. However, an evolutionarily related NHEJ apparatus has now been identified and characterized in the prokarya. Here we review the recent discoveries concerning bacterial NHEJ and discuss the possible origins of this repair system. We also examine the insights gained from the recent cellular and biochemical studies of this DSB-repair process and discuss the possible cellular roles of an NHEJ pathway in the life-cycle of prokaryotes and phages.     

Individual recognition: it is good to be different

Individual recognition (IR) behavior has been widely studied, uncovering spectacular recognition abilities across a range of taxa and modalities. Most studies of IR focus on the recognizer (receiver). These studies typically explore whether a species is capable of IR, the cues that are used for recognition and the specializations that receivers use to facilitate recognition. However, relatively little research has explored the other half of the communication equation: the individual being recognized (signaler). Provided there is a benefit to being accurately identified, signalers are expected to actively broadcast their identity with distinctive cues. Considering the prevalence of IR, there are probably widespread benefits associated with distinctiveness. As a result, selection for traits that reveal individual identity might represent an important and underappreciated selective force contributing to the evolution and maintenance of genetic polymorphisms.