A novel CFTR disease-associated mutation causes addition of an extra N-linked oligosaccharide

We have examined the influence of a novel missense mutation in the fourth extracytoplasmic loop (EL4) of CFTR detected in a patient with cystic fibrosis. This substitution (T908N) creates a consensus sequence (N X S/T) for addition of an N-linked oligosaccharide chain near the C-terminal end of EL4. Oligosaccharyl transferase generally does not have access to this consensus sequence if it is closer than about twelve amino acids from the membrane. However, the T908N site is used, even though it is within four residues of the predicted membrane interface and the oligosaccharide chain added binds calnexin, a resident chaperone of the ER membrane. The chloride channel activity of this variant CFTR is abnormal as evidenced by a reduced rate of ³⁶Cl^– efflux and a noisy single channel open state. This may reflect some displacement of the membrane spanning sequence C-terminal of EL4 since it contains residues influencing the ion pore.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na⁺/l-glutamate (l-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K⁺ and Cl^?. The presence of 100 mM K⁺ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K⁺ concentration and that in the absence of any sodium gradient, an outward K⁺ gradient was unable to influence the Na⁺/acidic amino acid transport system. It was also found that Cl^? could specifically activate the Na⁺-dependent l-glutamate (l-aspartate) uptake either in the presence or in the absence of K⁺. Also the effect of Cl^? was observed only in the presence of an inward Na⁺ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na⁺ gradient and in the presence of chloride gradient. l-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K⁺ or Cl^? did not show any translocation of net charge.     

Light-, pH- and uncoupler-dependent association of chloride with chloroplast thylakoids

Studies of the association of Cl^? with Photosystem (PS) II in CF₁-containing thylakoid membranes revealed that photosynthetically active Cl^? is retained in a Cl^?-free medium unless it is sufficiently alkaline, uncoupling conditions are established and light is excluded. After treatment under such conditions, electron transport from water became dependent on added Cl^? under all conditions. Quantitative measurements of ³⁶Cl^? retention in the light revealed that there were about five Cl^? anions present in Cl^?-sufficient chloroplasts per PS II reaction center, and one-fourth of that in Cl^?-deficient samples. Uncouplers representing three different types of uncoupling mechanism were found to be effective mediators of Cl^? release from thylakoids. Since the ability to collapse a proton gradient probably is the only property shared by all the tested uncouplers, a proton gradient may be involved in the retention of Cl^?. As uncoupler-mediated Cl^? release did not depend on preillumination of our samples, a long-lived proton gradient must exist in dark-adapted chloroplasts which may not span the whole thickness of the thylakoid membrane. It is postulated that the Cl^? active in PS II reactions resides in a special membrane domain from which protons slowly equilibrate with those in the bulk solutions. Cl^? is thought to be released to the bulk phases only when the pH of the membrane domain is raised above a certain threshold by the action of uncouplers. This domain may be identical to the intramembranous compartment which has been postulated to be associated with PS II (Prochaska, L.J. and Dilley, R.A., (1978) Front. Biol. Res. Energ. 1, 265–274).     

Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

Amino acid current through anion channels in cultured human glial cells

During volume regulation in hypotonic media, glial cells release a large portion of their amino acids. These amino acid losses appear to be mediated by a diffusion type of transport and a swelling-activated chloride channel seems to be involved. The objective of this project was to provide direct evidence that amino acids could diffuse through a Cl^? channel. Using a human glial cell line, Cl^? currents activated in hypotonic media were measured in whole-cell patch clamp. To measure the currents produced by amino acids, it was necessary to increase the pH of external solutions to basic values reaching 9.6 and 10.0 to raise the concentration of the anionic form of these amino acids. Introducing external hypotonic media containing high concentrations of amino acids, like glycine, taurine, glutamine and glutamate, it was possible to measure their respective current-voltage curves with NMDG-Cl-filled pipettes. From the reversal potentials, their permeability ratios with respect to chloride were determined. It was found that the low molecular weight amino acids, like glycine, were most permeant, while the larger ones, like glutamine, had a lower permeability with respect to chloride. The amino acids with two carboxyl groups, like glutamate, had a much lower permeability ratio. The reversal potentials for some metabolites, like lactate and malate were also measured for comparison. These results demonstrate that amino acids can diffuse through anion channels and that activation of these channels in pathological conditions could be at least partly responsible for the observed increase in external amino acids.     

Structure based computational assessment of channel properties of assembled ORF‐8a from SARS‐CoV

ORF 8a is a short 39 amino acid bitopic membrane protein encoded by severe acute respiratory syndrome causing corona virus (SARS‐CoV). It has been identified to increase permeability of the lipid membrane for cations. Permeability is suggested to occur due to the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides are generated using the first 22 amino acids, which include the transmembrane domain. Low energy structures reveal a hydrophilic pore mantled by residues Thr‐8, and ?18, Ser‐11, Cys‐13, and Arg‐22. Potential of mean force (PMF) profiles for mono (Na⁺, K⁺, Cl^?) and divalent (Ca²⁺) ions along the pore are calculated. The data support experimental findings of a weak cation selectivity of the channel. Calculations on 8a are compared to data derived for a pentameric bundle consisting of the M2 helices of the bacterial pentameric ligand gated ion channel GLIC (3EHZ). PMF curves of both, bundles 8a and M2, show sigmoidal shaped profiles. In comparison to the data for the M2‐GLIC model, data of the 8a bundle show lower amplitude of the PMF values between maximum and minimum and less discrimination amongst ions. Proteins 2015; 83:300–308. © 2014 Wiley Periodicals, Inc.     

The role of Cl<Superscript>−</Superscript> in pollen germination and tube growth

The involvement of Cl^? in cytoplasm polarization in the pollen tube and membrane potential control during pollen germination in vitro was studied by fluorescence techniques in Nicotiana tabacum. Cl^? release from cells was blocked by the anion channel inhibitor nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or by the addition of Cl^? to the incubation medium. The concentrations of the inhibitor (40 μM) and extracellular Cl^? completely inhibiting pollen germination (200 mM) and pollen tube growth (100 mM) were used. The release of anions from the pollen grain has been revealed in the first minutes of hydration also in the presence of 200 mM Cl^?. The inhibitor blocked this process completely, which points to the significance of the NPPB-sensitive anion channels in the transmembrane Cl^? transport at the early activation stage. The pollen tube membrane was hyperpolarized in the presence of 100 mM Cl^?; however, exogenous Cl^? had no effect on the compartmentalization and organelle movement in the tube. The inhibitor depolarized the plasma membrane in the pollen grain and tube and affected the polar organization of the cytoplasm and organelle movement. Thus, activity of NPPB-sensitive chloride channels was required to regulate the potential on the plasma membrane and to maintain the functional compartmentalization of the cytoplasm, which provides for the polar growth.     

Protein kinase A-regulated Cl− channel in ML-1 human hematopoietic myeloblasts

Using the inside-out patch clamp technique, we identified a Cl^? channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl^? channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (?40 mV). Biophysical studies of the Cl^? channel revealed that the current-voltage (I-V) relationship of the Cl^? channel was outwardly rectifying in symmetrical 142 mm Cl^? solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at ?60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl^? channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC⁵⁰) for 9-AC and DIDS were 174 ± 20 and 70±16 μm, respectively. Blockade of the Cl^? channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl^? channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses.     

Molecular dynamics of water and monovalent‐ions transportation mechanisms of pentameric sarcolipin

The Sarcolipin (SLN) is a transmembrane protein that can form a self‐assembled pentamer. In this work, the homology modeling and all‐atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na⁺, Cl^? and water molecule along the pore channel of penta‐SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na⁺ and Cl^? could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na⁺ and Cl^? could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu²¹ that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na⁺ and Cl^? ion permeability by hydrated and vacuum states. Proteins 2016; 84:73–81. © 2015 Wiley Periodicals, Inc.     

Theoretical studies on protein-nucleic acid interactions. II. Hydrogen bonding of amino acid side chains with bases and base pairs of nucleic acids

With a view to understanding the role of hydrogen bonds in the recognition of nucleic acids by proteins, hydrogen bonding between the bases and base pairs of nucleic acids and the amino acids (Asn, Gln, Asp and Glu, and charged residues Arg⁺, Glu^?, and Asp^?) has been studied by a second-order perturbation theory. Binding energies have been calculated for all possible configurations involving a pair of hydrogen bonds between the base (or base pair) and the amino acid residue. Our results show that the hydrogen bonding in these cases has a large contribution from electrostatic interaction. In general, the charged amino acids, compared to the uncharged ones, form more stable complexes with bases or base pairs. The hydrogen-bond energies are an order of magnitude smaller than the Coulombic interaction energies between basic amino acids (Lys⁺, Arg⁺, and His⁺) and the phosphate groups of nucleic acids. The stabilities of the complexes of amino acids Asn, Gln, Asp, and Glu with bases are in the order: G–X > C–X > A–X U–X or T–X, and G · C–X > A · T(U)–X, where X is one of these amino acid residues. It has been shown that Glu^? and Asp^? can recognize guanine in single-stranded nucleic acids; Arg⁺ can recognize G · C base pairs from A · T base pairs in double-stranded structures.     

Involvement of different S4 parts in the voltage dependency of Na channel gating

Summary Three synthetic peptides corresponding to parts of S₄ of the first repeat of eel electroplax sodium channel were synthesized. The basic peptide was C ₁ ⁺ which corresponds to amino acids 210–223 (eel channel numbering) and two subfractions: an external fraction, C _1ex ⁺ (amino acid 210–217); and an internal part, C _1in ⁺ (amino acid 218–221). Peptide C ₁ ⁺ includes four of the charged amino acids of this domain; peptide C _1ex ⁺ includes three of the charged amino acids and is closer to the external membrane surface (according to channel models) than peptide C _1in ⁺ which includes the fourth charged amino acid alone.Antibodies generated in rabbits against these peptides were shown to be site specific. Using the whole-cell patch-clamp technique, we found that in rat dorsal root ganglion (DRG) cells, the antibodies against C _1in ⁺ but not against C _1ex ⁺ had an effect on the gating parameters. They shifted the Na-channel inactivation curve towards hyperpolarization and decreased the slope of the Na-channel activation curve. These results demonstrate that during the conformational changes associated with channel gating, the fourth charged amino acid of S₄ must be accessible to antibodies given to the external solution. Furthermore, they indicate a specific involvement of S₄ in the voltage dependency of the gating processes.This study was supported by a basic research grant of The Israel Academy of Sciences and Humanities (#430.87 to H.M. and G.S.).We wish to express our gratitude to Dr. M. Tosteson (Harvard Medical School) for providing us with samples of peptide S₄IV to use in the ELISA assays. We thank Dr. R. Gordon (The Max Planck Institute for Biophysics, Frankfurt) for immunochemical advise and protocols. The advice of Drs. M. Sammar, M. Paizi, R. Schatzberger, I. Zeitoun and Y. Mika (Technion) was very useful. We thank Mrs. A. Schwartz (Technion) for participating in the experiments.     

Functional Swapping between Transmembrane Proteins TMEM16A and TMEM16F

The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca²⁺-dependent Cl⁻ ion transport, and TMEM16F is responsible for Ca²⁺-dependent phospholipid scrambling. Here we established assay systems for the Ca²⁺-dependent Cl⁻ channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F^−/− immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl⁻ channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl⁻ channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.     

Energy sources for amino acid transport in animal cells

The existence of an electrogenic Na⁺ pump in Ehrlich cells which substantially contributes to the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP⁺), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondrial membrane potential, by adding to the overall potential as measured in this way is likely to cause an overestimation of the membrane potential difference (p.d.). But since this error tends to diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of an adequate Na⁺ concentration gradient. Accordingly, the F₂-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP⁺ at constant Na⁺ concentrations, suggesting that each responds directly to membrane potential. There is evidence that the electrogenic (free) movement of Cl^? is slow, at least at normal p.d., whereas a major part of the Cl^? movement across the cellular membrane appears to occur by an electrically silent Cl^?-base exchange mechanism. By such a mode Cl^?, together with an almost stoichiometric amount of K⁺, may under certain conditions move into the cell against a high adverse electrical potential difference. This “paradoxical” movement of K⁺Cl^? contributing to the deviation of the Cl^? distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide. As a likely explanation a H⁺–K⁺ exchange pump was previously offered, even though unequivocal evidence of such a pump is so far lacking. According to available evidence the electrogenic movement of free Cl^? is too small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. The evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na⁺ concentration gradient, the electrogenic Na⁺ pump will contribute sufficient extra driving force to actively transport amino acid into the cells.     

A major role for chloride in (3H)- noradrenaline transport by rat heart adrenergic nerves

The effect of Cl^? and other anions on (³H)-noradrenaline line (NA) transport by bisected rat heart atrial appendages $\text{in}$$\text{vitro}$ has been studied. It was found that NA active transport, at the plasma membrane level, shows an absolute dependency on Cl^?, with a half-maximal activation of transport occurring at 6 mM Cl^? and complete saturation at 50 mM. Cl^? effects are due to changes in transport Km, while Vmax is not changed. Only one class of sites for Cl^? seem to be present in the transport system. Br^? can substitute for Cl^? with 90% effectiveness, nitrate and iodide are less effective, while larger anions are very poor substitutes. In addition, heart atrial hemi-appendages have been characterized as a suitable preparation for studies of this type.     

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl^?) channels. The EAATs couple the transport of glutamate to the co-transport of three Na⁺ ions and one H⁺ ion into the cell, and the counter-transport of one K⁺ ion out of the cell. The EAAT Cl^? channel is activated by the binding of glutamate and Na⁺, but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, Glt_Ph, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl^? permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl^? permeation and the mechanisms that underlie their split personality.     

Summary

The plasma membrane H⁺-ATPase in higher plants has been implicated in nutrient uptake, phloem loading, elongation growth and establishment of turgor. Although a C-terminal regulatory domain has been identified, little is known about the physiological factors involved in controlling the activity of the enzyme. To identify components which play a role in the regulation of the plant H⁺-ATPase, a fusicoccin responsive yeast expressing Arabidopsis plasma membrane H⁺-ATPase AHA2 was employed. By testing the fusicoccin binding activity of yeast membranes, the C-terminal regulatory domain of AHA2 was found to be part of a functional fusicoccin receptor, a component of which was the 14–3-3 protein. ATP hydrolytic activity of AHA2 expressed in yeast internal membranes was activated by all tested isoforms of the 14–3-3 protein of yeast and Arabidopsis, but only in the presence of fusicoccin, and activation was prevented by a phosphoserine peptide representing a known 14–3-3 protein binding motif in Raf-1. The results demonstrate that the 14–3-3 protein is an activator molecule of the H⁺-ATPase and provides the first evidence of a protein involved in activation of plant plasma membrane H⁺-ATPase.     

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.     

Frog Virus 3 Open Reading Frame 97R Localizes to the Endoplasmic Reticulum and Induces Nuclear Invaginations

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.     

Characterization of an Atypical Member of the Na+/Cl−-Dependent Transporter Family: Chromosomal Localization and Distribution in GABAergic and Glutamatergic Neurons in the Rat Brain

Abstract: A 3.7-kb cDNA fragment, designated rat-XT1, was isolated from a rat whole-brain cDNA library. The nucleotide sequence of XT1 codes for a 727 amino acid protein with a calculated molecular mass of 81,139 Da and 12 putative transmembrane domains. This protein shares significant homology (28–32%) with the monoamine- (dopamine, norepinephrine, serotonin), amino acid- (taurine, proline, GABA, glycine), choline-, and betaine-, Na⁺/Cl^?-dependent transporters. The homology is especially high within the first, second, sixth, and eighth transmembrane domains (45–75%). Thus, XT1 clearly belongs to the Na⁺/Cl^?-dependent neurotransmitter transporter superfamily. However, XT1 may define a new subfamily of transporter because it differs structurally from other members of this family in that the extracellular loop linking transmembrane domains 7 and 8 and the C-terminal tail are significantly larger in size. Transient or stable expression of rat-XT1 failed to confer to the transfected cells the ability to transport actively any of the >60 established or putative neurotransmitter substances assessed. Northern blot analyses of peripheral and neural tissues demonstrated that expression of the 8-kb XT1 mRNA is essentially restricted to the nervous system. In situ hybridization demonstrated a broad but discrete localization of XT1 message in the CNS, particularly in the cerebellum (Purkinje and granular cell layers), the hippocampus (pyramidal and granular cell layers), and the thalamus and throughout the cerebral cortex. This distribution parallels that of the neurotransmitters glutamate and aspartate; however, neither of these excitatory amino acids is a substrate for transport. One noticeable exception to the codistribution of the mRNA for rat-XT1 and these excitatory neurotransmitters is the cerebellar Purkinje cell layer, in which GABAergic neurons are localized. The gene encoding for XT1 is localized to the mouse chromosome 3 in the vicinity of the locus for the mouse neurological disorder spastic (spa).