Search

The potential for relapse following cessation of drug use can last for years, implying the induction of stable changes in neural circuitry. In hippocampal slices from rats treated with nicotine for 1 week, withdrawal from nicotinein vivoproduces an increase in CA1 pyramidal cell excitability that persists up to 9 months. Immediately upon drug cessation, the enhanced excitability depends on input from regions upstream of CA1, while the long-term excitability change (> 4 weeks) is expressed as an increase in the intrinsic excitability of CA1 neurons. Re-exposure to nicotinein vitrorestores hippocampal function to control levels via activation of high-affinity nicotinic acetylcholine receptors after 1 d of withdrawal, but not at times >4 weeks. Thus, nicotinein vivofirst induces homeostatic adaptations followed by other more robust neural changes. These mechanisms may contribute to hippocampal localized cue-motivated reinstatement of drug-seeking and/or cognitive deficits observed during withdrawal.

The mouse gamma-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [(3)H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [(3)H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented approximately 30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

The uptake of neurotransmitter by plasma membrane transporters is a principal method for regulating extracellular transmitter levels. Neurotransmitter-mediated signals in turn are able to regulate transporter expression and function. Thus, there is a continual interplay between transporters and the transmitters they transport. Previously we showed that extracellular gamma-aminobutyric acid (GABA) increases the expression of the GABA transporter 1 (GAT1) on a time scale of minutes by acting via the transporter to slow transporter internalization. This mechanism requires in part direct tyrosine phosphorylation of the transporter. In the present study we show that the presence of GABA on a longer time scale causes a net decrease in GAT surface expression. The decrease in expression represents the contributions of transporter-mediated up-regulation and a more substantial GABA-receptor-mediated down-regulation. This receptor-mediated down-regulation is the result of both changes in the rates of transporter trafficking and in the number of transporters available for trafficking. As with transporter-mediated regulation of GAT1, the receptor-mediated regulation is associated with changes in the direct phosphorylation of GAT1. These data suggest that multiple pathways, perhaps converging upon mechanisms involving protein phosphorylation, act to regulate GAT1 expression in neurons.

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons. This trafficking may be important in regulating neuronal signaling. Such regulation will be subject to the number of trafficking transporters and their trafficking rates. In the present study, we define an acutely recycling pool of endogenous γ-aminobutyric acid transporters (GAT1) in cortical neurons that comprises approximately one-third of total cellular GAT1. Kinetic analysis of this pool estimates exocytosis and endocytosis time constants of 1.6 and 0.9 min, respectively, and thus approximately one-third of the recycling pool is plasma membrane resident in the basal state. Recent evidence shows that GAT1 substrates, second messengers, and interacting proteins regulate GAT1 trafficking. These triggers could act by altering trafficking rates or by changing the recycling pool size. In the present study we examine three GAT1 modulators. Calcium depletion decreases GAT1 surface expression by diminishing the recycling pool size. Sucrose increases GAT1 surface expression by blocking clathrin- and dynamin-dependent endocytosis, but it does not change the recycling pool size. Protein kinase C decreases surface GAT1 expression by increasing the endocytosis rate, but it does not change the exocytosis rate or the recycling pool size. Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that ∼1000 transporters comprise the acutely recycling pool, of which 300 are on the surface in the basal state, and five transporters insert into the plasma membrane every second. This insertion could represent the fusion of one transporter-containing vesicle.

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins.

Serotonin transporters (SERTs), sites of psychostimulant action, display multiple conducting states in expression systems. These include a substrate-independent transient conductance, two separate substrate-independent leak conductances associated with Na + and H + , and a substrate-dependent conductance of variable stoichiometry, which exceeds that predicted from electroneutral substrate transport. The present data show that the SNARE protein syntaxin 1A binds the N-terminal tail of SERT, and this interaction regulates two SERT-conducting states. First, substrate-induced currents are absent because Na + flux becomes strictly coupled to 5HT transport. Second, Na + -mediated leak currents are eliminated. These two SERT-conducting states are present endogenously in thalamocortical neurons, act to depolarize the membrane potential, and are modulated by molecules that disrupt SERT and syntaxin 1A interactions. These data show that protein interactions govern SERT activity and suggest that both cell excitability and psychostimulant-mediated effects will be dependent upon the state of association among SERT and its interacting partners.

GABA transporters control extracellular GABA levels by coupling transmitter uptake to the sodium and chloride cotransport. The rat brain GABA transporter GAT1 and other members of this family are regulated by direct interactions with syntaxin 1A, a protein involved in vesicle docking and in the regulation of several ion channels and transporters. We have shown previously that syntaxin 1A exerts its effects on GAT1 by decreasing the net uptake of GABA and its associated ions through interactions with aspartic acid residues in the N-terminal tail of GAT1. This reduction in net uptake could be caused by many steps in the transport cycle, including substrate binding, substrate flux, substrate efflux, or reorientation of the unliganded transporter. To address this question, we performed GABA flux assays, measured flux- and efflux-associated ion currents, and assessed GABA exchange in multiple experimental systems expressing syntaxin 1A and wild-type GAT1 or GAT1 mutants. Syntaxin 1A caused similar reductions in forward and reverse transport that did not involve changes in apparent transport affinities for sodium, chloride, or GABA. The syntaxin 1A-mediated reduction in GABA flux and efflux was mimicked by mutations in GAT1 at the syntaxin 1A binding site. The binding site GAT1 mutant also caused a reduction in exchange. These data suggest that syntaxin 1A exerts its effects, directly or indirectly, on GAT1 function through interactions with GAT1's N-terminal tail and that the inhibition occurs at a step in the translocation process after substrate binding but which involves both unidirectional transport and transmitter exchange.

Atropine, a classical muscarinic antagonist, has been reported previously to inhibit neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, the action of atropine has been examined on α3β4 receptors expressed heterologously in Xenopus oocytes and native nAChRs in medial habenula neurons. At concentrations of atropine often used to inhibit muscarinic receptors (1 μM), responses induced by near-maximal nicotine concentrations (100 μM) at negative holding potentials (−65 mV) are inhibited (14–30%) in a reversible manner in both α4 and α3 subunit-containing heteromeric nAChRs. Half-maximal effective concentrations (IC50 values) for atropine inhibition are similar for the four classes of heteromeric receptors studied (4–13 μM). For α3β4 nAChRs in oocytes, inhibition by atropine (10 μM) is not overcome at higher concentrations of agonist, and is increased with membrane hyperpolarization. These results are consistent with non-competitive antagonism – possibly ion channel block. At low concentrations of both nicotine (10 μM) and atropine (

Plasma membrane neurotransmitter transporters affect synaptic signaling through transmitter sequestration. Transporters redistribute to and from the plasma membrane, suggesting a role for trafficking in regulating synaptic transmitter levels. One method for controlling transmitter levels would be to regulate transporter redistribution in parallel with transmitter release. Thus, how similar are these processes? We show that the trafficking of the GABA transporter GAT1 resembles the trafficking of neurotransmitter-filled synaptic vesicles: (1) transporters located on the plasma membrane are internalized and reinserted into the plasma membrane on the order of minutes; (2) the rate of recycling is depolarization and calcium dependent; (3) GAT1 internalization is associated with clathrin and dynamin; and (4) intracellular GAT1 is associated with multiple compartments and, more importantly, is found on a distinct class of vesicles. These vesicles are clear, ∼50 nm in diameter, and contain many proteins found on neurotransmitter-containing small synaptic vesicles; however, they appear to lack several traditional small synaptic vesicle proteins, such as synaptophysin and the vesicular GABA transporter. These data provide additional support for the hypothesis that GABA transporters traffic in parallel with neurotransmitter-containing small synaptic vesicles and also raise the possibility that some fraction of vesicles found in GABAergic neurons may not be participating in transmitter release but rather in the rapid regulated redistribution of membrane proteins involved in transmitter uptake.

The molecular mechanisms underlying polarized sorting of proteins in neurons are poorly understood. Here we report the identification of a 16 amino-acid, dileucine-containing motif that mediates dendritic targeting in a variety of neuronal cell types in slices of rat brain. This motif is present in the carboxy (C) termini of Shal-family K+ channels and is highly conserved from C. elegans to humans. It is necessary for dendritic targeting of potassium channel Kv4.2 and is sufficient to target the axonally localized channels Kv1.3 and Kv1.4 to the dendrites. It can also mediate dendritic targeting of a non-channel protein, CD8.

Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and synaptic Ca(2+) channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca(2+) channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca(2+) channel regulation by this t-SNARE.

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

Background. The regulation of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis. The effect of cyclic AMP (cAMP) on ENaC activity is tissue-specific, and is controversial when ENaC is ex-pressed in Xenopus oocytes. Methods. The modulation of ENaC by cAMP in oocytes expressing human or rat ENaC was performed with two-electrode voltage clamping. Results. 250 μM 8-(4-chlorophenylthio)-adenosine 3′, 5′-cyclic monophosphate (8-CPT-cAMP) added to the bath significantly increased normalized amiloride-sensitive currents within 60 s in oocytes expressing human α, β, and γ subunits (5 ng cRNA each). The cAMP effect was dose-dependent and was partially inhibited by 200 μM Rp-CPT-cAMP, a competitive cAMP antagonist. A transient effect of 8-CPT-cAMP on rat ENaC activity was also observed. Oocytes expressing rat α subunits with γ subunits (which have a putative protein kinase A phosphorylation site) showed similar increases in amiloride-sensitive current with 250 μM 8-CPT-cAMP, while oocytes expressing rat α subunits with β subunits were not activated by 8-CPT-cAMP. Further, rat ENaC (but not human ENaC)-expressing oocytes were not activated by cAMP when oocytes were continuously superfused during electrophysiological recordings, suggesting that rat ENaC activation by cAMP is dependent upon the condition of oocytes during cAMP stimulation. Conclusion. The present results suggest that ENaC expressed in Xenopus oocytes can be activated by cAMP, and that the γ subunit confers sensitivity to cAMP modulation of rat ENaC activity.

Pregabalin [(S)-(+)-3-isobutylgaba] and gabapentin [1-(aminomethyl)cyclohexane acetic acid] are γ-aminobutyric acid (GABA) derivatives that are effective in the treatment of behavioural disorders, convulsions, epilepsy and hyperalgesia. The mechanisms underlying the diverse actions of these compounds in the brain have not been well elucidated. To test the hypothesis that these compounds exert some of their effects on GABA-ergic systems in the brain, we examined their role in regulating the rat brain GABA transporter GAT1, a plasma membrane protein involved in regulating synaptic transmitter levels. Prolonged incubation of hippocampal cultures, which endogenously express GAT1, with gabapentin and pregabalin caused a 2-fold increase in subsequent GABA uptake, which was concentration- and time-dependent. This increase in uptake was correlated with a redistribution of GAT1 protein from intracellular locations to the plasma membrane. Further experiments also suggested that the signal transduction cascade that modulates pregabalin-mediated GAT1 redistribution may involve pathways activated by specific GAT1 substrates and antagonists but does not involve protein kinase C and tyrosine kinases, two other pathways known to regulate GAT1 redistribution. These data suggest that pregabalin and gabapentin may exert some of their actions in the brain by altering GABAergic signalling.

Neurotransmitter transporters couple the transport of transmitter against its concentration gradient to the electrochemical potential of associated ions which are also transported. Recent studies of some neurotransmitter transporters show them to have properties of both traditional carriers and substrate-dependent ion channels, in that ion fluxes are in excess of that predicted from stoichiometric substrate fluxes. Whether these properties are comparable for all transporters, the extent to which these permeation states are independent, and whether the relationship between these two states can be regulated are not well understood. To address these questions, we expressed the Drosophila serotonin (5HT) transporter (dSERT) in Xenopus oocytes and measured both substrate-elicited ion flux and 5HT flux at various temperatures and substrate concentrations. We find that the ion flux and 5HT flux components of the transport process have a significant temperature dependence suggesting that ion flux and transmitter flux arise from a similar thermodynamically-coupled process involving large conformational changes (e.g., gating). These data are in contrast to those shown for glutamate transporters, suggesting a different permeation process for 5HT transporters. The relationship between ion flux and 5HT flux is differentially regulated by chloride and 5HT, suggesting that these permeation states are distinct. The difference in half-maximal 5HT concentration necessary to mediate ion flux and 5HT flux occurs at submicromolar 5HT concentrations suggesting that the relative participation of dSERT in ion flux and 5HT flux will be determined by the synaptic 5HT concentration.

Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which appear to operate in order to maintain appropriate levels of transmitter in the synaptic cleft. One such mechanism is the trafficking of the transporter in association with synaptic vesicle release machinery. This report examines the specifics of trafficking regulation of the rat brain GABA transporter GAT1 by syntaxin 1A, a plasma membrane component of the SNARE complex which is involved in vesicle membrane fusion. In hippocampal neurons, botulinum neurotoxin 1C, which specifically cleaves syntaxin 1A, down-regulates plasma membrane GAT1 levels as assessed by surface biotinylation, suggesting that syntaxin 1A is a positive regulator of GAT1 surface expression. The up-regulation correlates with a decrease in intracellular GAT1 levels and is complete within several minutes. These data suggest that syntaxin 1A mediates the redistribution of GAT1 on a time scale important for the rapid regulation of extracellular GABA levels. Expression of different syntaxin 1A constructs in Xenopus oocytes suggests that several portions of the syntaxin 1A molecule are required for the trafficking of GAT1. These data suggest that the trafficking of GAT1 will be subject to regulatory control by the many molecules known to interact with various domains of syntaxin 1A.

Relationships between nicotinic acetylcholine receptor (nAChR) channel function and nAChR subunit mRNA expression were explored in acutely isolated rat medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT-PCR techniques. Following amplification using subunit-specific primers, subunits could be categorized in one of three ways: (i) present in 95-100% cells: alpha3, alpha4, alpha5, beta2 and beta4; (ii) never present: alpha2; and (iii) sometimes present ( approximately 40% cells): alpha6, alpha7 and beta3. These data imply that alpha2 subunits do not participate in nAChRs on MHb cells, that alpha6, alpha7 and beta3 subunits are not necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of alpha3, alpha4, alpha5, beta2 and beta4 subunits. Little difference in the patterns of subunit expression between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may be an insufficient determinant of nAChR channel function. Normalization of nAChR subunit levels to the amount of actin mRNA, however, revealed that cells with functional channels were associated with high levels (0.78 relative to actin; 11/12 cells) of all of the category (i) subunits: alpha3, alpha4, alpha5, beta2 and beta4. Conversely, one or more of these subunits was always low (0.40 relative to actin) in all cells with no detectable response to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs.

Chloride (Cl−) homeostasis is critical for many cell functions including cell signaling and volume regulation. The action of GABA at GABAAreceptors is primarily determined by the concentration of intracellular Cl−. Developmental regulation of intracellular Cl−results in a depolarizing response to GABA in immature neocortical neurons and a hyperpolarizing or shunting response in mature neocortical neurons. One protein that participates in Cl−homeostasis is the neuron-specific K+–Cl−cotransporter (KCC2). Thermodynamic considerations predict that in the physiological ranges of intracellular Cl−and extracellular K+concentrations, KCC2 can act to either extrude or accumulate Cl−. To test this hypothesis, we examined KCC2 function in pyramidal cells from rat neocortical slices in mature (18–28 d postnatal) and immature (3–6 d postnatal) rats. Intracellular Cl−concentration was estimated from the reversal potential of whole-cell currents evoked by local application of exogenous GABA. Both increasing and decreasing the extracellular K+concentration resulted in a concomitant change in intracellular Cl−concentration in neurons from mature rats. KCC2 inhibition by furosemide caused a change in the intracellular Cl−concentration that depended on the concentration of pipette Cl−; in recordings with low pipette Cl−, furosemide lowered intracellular Cl−, whereas in recordings with elevated pipette Cl−, furosemide raised intracellular Cl−. In neurons from neonatal rats, manipulation of extracellular K+had no effect on intracellular Cl−concentration, consistent with the minimal KCC2 mRNA levels observed in neocortical neurons from immature animals. These data demonstrate a physiologically relevant and developmentally regulated role for KCC2 in Cl−homeostasis via both Cl−extrusion and accumulation.

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.

The gene for a β-neurotoxin [ Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [ 3 H]GABA [γ-aminobutyric acid (GABA)] in neuronal cells as effectively as natural β-toxins.

Plasma membrane neurotransmitter transporters are a family of integral membrane proteins, found on both neurons and glia, that have the capacity to influence neuronal signaling through a number of mechanisms including transmitter reuptake and ionic flux. Clinically, these proteins are of interest because their dysfunction is associated with several neurological and psychiatric disorders, and because they are the targets of many drugs of abuse and therapy. In this review, the authors focus on one of the more recent, fascinating discoveries about neurotransmitter transporters; namely, that transporter function is regulated by altering the number of transporters on the cell surface. These data suggest that transporter expression is in continual flux and that transporters respond to their environment in an effort to maintain baseline transmitter levels in the brain. The authors examine the mechanisms underlying changes in transporter number, discuss clinical disorders that are correlated with transporter expression, and suggest that controlling transporter redistribution may be a future therapeutic strategy for disorders related to abnormal transmitter levels.

It is hypothesized that desensitization of neuronal nicotinic acetylcholine receptors (nAChRs) induced by chronic exposure to nicotine initiates upregulation of nAChR number. To test this hypothesis directly, oocytes expressing α4β2 receptors were chronically incubated (24–48 hr) in nicotine, and the resulting changes in specific [3H]nicotine binding to surface receptors on intact oocytes were compared with functional receptor desensitization. Four lines of evidence strongly support the hypothesis. (1) The half-maximal nicotine concentration necessary to produce desensitization (9.7 nm) was the same as that needed to induce upregulation (9.9 nm). (2) The concentration of [3H]nicotine for half-maximal binding to surface nAChRs on intact oocytes was also similar (11.1 nm), as predicted from cyclical desensitization models. (3) Functional desensitization of α3β4 receptors required 10-fold higher nicotine concentrations, and this was mirrored by a 10-fold shift in concentrations necessary for upregulation. (4) Mutant α4β2 receptors that do not recover fully from desensitization, but not wild-type channels, were upregulated after acute (1 hr) applications of nicotine. Interestingly, the nicotine concentration required for half-maximal binding of α4β2 receptors in total cell membrane homogenates was 20-fold lower than that measured for surface nAChRs in intact oocytes. These data suggest that cell homogenate binding assays may not accurately reflect thein vivodesensitization affinity of surface nAChRs and may account for some of the previously reported differences in the efficacy of nicotine for inducing nAChR desensitization and upregulation.

gamma-Aminobutyric acid (GABA) transporters on neurons and glia at or near the synapse function to remove GABA from the synaptic cleft. Recent evidence suggests that GABA transporter function can be regulated, although the initial triggers for such regulation are not known. One hypothesis is that transporter function is modulated by extracellular GABA concentration, thus providing a feedback mechanism for the control of neurotransmitter levels at the synapse. To test this hypothesis, GABA uptake assays were performed on primary dissociated rat hippocampal cultures that endogenously express GABA transporters and on mammalian cells stably expressing the cloned rat brain GABA transporter GAT1. In both experimental systems, extracellular GABA induces chronic changes in GABA transport that occur in a dose-dependent and time-dependent manner. In addition to GABA, ACHC and nipecotic acid, both substrates of GAT1, up-regulate transport; GAT1 transport inhibitors that are not transporter substrates down-regulate transport. These changes occur in the presence of blockers of both GABAA and GABAB receptors, occur in the presence of protein synthesis inhibitors, and are not influenced by intracellular GABA. Surface biotinylation experiments reveal that the increase in transport is correlated with an increase in surface transporter expression. This increase in surface expression is due, at least in part, to a slowing of GAT1 internalization in the presence of extracellular GABA. These data suggest that the GABA transporter fine-tunes its function in response to extracellular GABA and would act to maintain a constant level of neurotransmitter at the synaptic cleft.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist- induced, Ca(2+)-activated Cl- currents that were measured using a two- electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX- sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor- activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

We report that activators and inhibitors of protein kinase C (PKC) and protein phosphatases regulate the activity of a cloned rat brain gamma- aminobutyric acid (GABA) transporter (GAT1) expressed in Xenopus oocytes. Four compounds known to activate PKC increased GABA uptake 2- 3.5-fold over basal control levels. Inhibition of PKC by bisindolylmaleimide reduced basal GABA uptake 80% and blocked the phorbol 12-myristate 13-acetate (PMA)-induced stimulation of transport. Okadaic acid, a protein phosphatase inhibitor, stimulated transport 2.5- fold; a 4-fold increase in GABA uptake occurred when oocytes were treated with cyclosporin A, a specific inhibitor of protein phosphatase 2B. Modulation resulted in changes to Vmax but not to Km and was influenced by the functional expression level of the transporter protein; as expression level increased, the ability to up-regulate transporter activity decreased. Down-regulation of transporter activity was independent of expression level. Modulation did not occur through phosphorylation of the three consensus PKC sites predicted by the primary protein sequence since their removal had no effect on the susceptibility of the transporter to modulation by PMA or bisindolylmaleimide. Subcellular fractionation of oocyte membranes demonstrated that under basal level conditions, the majority of GAT1 was targeted to a cytoplasmic compartment corresponding to the trans- Golgi or low density vesicles. Stimulation of PKC with PMA resulted in a translocation of transporters from this compartment to the plasma membrane. At higher expression levels of GAT1 protein, a larger portion of GAT1 was found on the plasma membrane during basal level conditions and treatment with bisindolylmaleimide resulted in removal of these transporters from the plasma membrane. At expression levels demonstrated to be resistant to modulation by PMA, PMA-treatment still resulted in translocation of transporters from the cytoplasm to the plasma membrane. Thus, the inability of PMA to increase uptake at high expression of the GAT1 protein is due to saturation at a step subsequent to translocation. These findings 1) demonstrate the presence of a novel regulated secretory pathway in oocytes and 2) suggest a modulatory mechanism for neurotransmitter transporters that could have significant effects upon synaptic function.

The neurotransmitter transporters belong to three known families of intrinsic membrane proteins (Figure 1). The energy for transport, which is often against the neurotransmitter concentration gradient, is derived from the cotransport (and in some cases the counter transport) of inorganic ions. Ion-transporting ATPases establish the concentration gradients for these ions.

Nucleotide binding properties of the G protein alpha subunit G(o)alpha were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTP gamma S (guanosine 5'-(3-O-thio)-triphosphate) binding on the colony lifts following transformation of bacteria with modified G(o)alpha cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of G(o)alpha for GTP gamma S or Mg2+ were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in p21ras proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP gamma S binding but retained the capacity to interact with the beta gamma subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type G(o)alpha increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced Cl- response, suggesting that this mutation resulted in a dominant negative phenotype.

Plasma membrane neurotransmitter transporters are regulators of extracellular transmitter levels in brain and are the primary sites of action for several drugs of abuse and therapy. Studies are beginning to reveal how neurons use synaptic machinery to modulate these regulators.

Plasma membrane serotonin transporters (SERTs) regulate serotonin (5HT) levels in brain and are a site of action of antidepressants and psychostimulant drugs of abuse. Syntaxin 1A is a component of the synaptic vesicle docking and fusion apparatus and has been shown to interact with multiple plasma membrane neurotransmitter transporters including SERT. Previously, we showed that syntaxin 1A regulates the transport stoichiometry of SERT. When not bound to syntaxin 1A, SERT shows both substrate-independent Na(+) fluxes and substrate-dependent Na(+) fluxes of variable stoichiometry; these fluxes are eliminated in the presence of syntaxin 1A as Na(+) flux becomes strictly coupled to 5HT uptake. However, not known are the endogenous signaling molecules that determine the conducting states that SERT exhibits. In the present experiments, we show that inhibitors of calcium/calmodulin-dependent kinase II (CaM kinase II) modulate the stoichiometry of 5HT flux and that this effect requires syntaxin 1A. The modulation correlates with a shift in the affinity of SERT for syntaxin 1A binding. The regulation by CaM kinase II is eliminated by a mutation in the N-terminal domain of SERT. In neonatal thalomocortical neurons that endogenously express SERT and syntaxin 1A, inhibition of CaM kinase II reveals SERT-mediated currents. These data suggest that calcium-mediated signals can serve as a trigger for regulating protein-protein interactions that control SERT conducting states.

The SNARE hypothesis of vesicle fusion proposes that a series of protein-protein interactions governs the delivery of vesicles to various membrane targets such as the Golgi network and the plasma membrane. Key players in this process include members of the syntaxin family of membrane proteins. The first member identified in this family, syntaxin 1A, plays an essential role in the docking and fusion of neurotransmitter-containing vesicles to the presynaptic membrane of neurons. Syntaxin 1A and other syntaxin family members have also been shown to interact with, and directly regulate, a variety of ion channels. More recently, the family of plasma membrane neurotransmitter transporters, proteins that function in part to control transmitter levels in brain, have been shown to be direct targets of syntaxin 1A regulation. This regulation involves both the trafficking of transporters as well as the control of ion and transmitter flux through transporters. In this chapter, the functional effects of syntaxin-transporter interactions are reviewed, and how such interactions may regulate neuronal signaling are considered.

Sodium-dependent neurotransmitter transporters participate in the clearance and/or recycling of neurotransmitters from synaptic clefts. The snf-11 gene in Caenorhabditis elegans encodes a protein of high similarity to mammalian GABA transporters (GATs). We show here that snf-11 encodes a functional GABA transporter; SNF-11–mediated GABA transport is Na+and Cl−dependent, has an EC50value of 168 μM, and is blocked by the GAT1 inhibitor SKF89976A. The SNF-11 protein is expressed in seven GABAergic neurons, several additional neurons in the head and retrovesicular ganglion, and three groups of muscle cells. Therefore, all GABAergic synapses are associated with either presynaptic or postsynaptic (or both) expression of SNF-11. Although a snf-11 null mutation has no obvious effects on GABAergic behaviors, it leads to resistance to inhibitors of acetylcholinesterase. In vivo, a snf-11 null mutation blocks GABA uptake in at least a subset of GABAergic cells; in a cell culture system, all GABA uptake is abolished by the snf-11 mutation. We conclude that GABA transport activity is not essential for normal GABAergic function in C. elegans and that the localization of SNF-11 is consistent with a GABA clearance function rather than recycling.

α7 nicotinic acetylcholine receptors (nAChRs) modulate network activity in the CNS. Thus, functional regulation of α7 nAChRs could influence the flow of information through various brain nuclei. It is hypothesized here that these receptors are amenable to modulation by tyrosine phosphorylation. In bothXenopusoocytes and rat hippocampal interneurons, brief exposure to a broad-spectrum protein tyrosine kinase inhibitor, genistein, specifically and reversibly potentiated α7 nAChR-mediated responses, whereas a protein tyrosine phosphatase inhibitor, pervanadate, caused depression. Potentiation was associated with an increased expression of surface α7 subunits and was not accompanied by detectable changes in receptor open probability, implying that the increased function results from an increased number of α7 nAChRs. SolubleN-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis was shown to be a plausible mechanism for the rapid delivery of additional α7 nAChRs to the plasma membrane. Direct phosphorylation/dephosphorylation of α7 subunits was unlikely because mutation of all three cytoplasmic tyrosine residues did not prevent the genistein-mediated facilitation. Overall, these data are consistent with the hypothesis that the number of functional cell surface α7 nAChRs is controlled indirectly via processes involving tyrosine phosphorylation.

Plasma membrane neurotransmitter transporters rapidly traffic to and from the cell surface in neurons. This trafficking may be important in regulating neuronal signaling. Such regulation will be subject to the number of trafficking transporters and their trafficking rates. In the present study, we define an acutely recycling pool of endogenous gamma-aminobutyric acid transporters (GAT1) in cortical neurons that comprises approximately one-third of total cellular GAT1. Kinetic analysis of this pool estimates exocytosis and endocytosis time constants of 1.6 and 0.9 min, respectively, and thus approximately one-third of the recycling pool is plasma membrane resident in the basal state. Recent evidence shows that GAT1 substrates, second messengers, and interacting proteins regulate GAT1 trafficking. These triggers could act by altering trafficking rates or by changing the recycling pool size. In the present study we examine three GAT1 modulators. Calcium depletion decreases GAT1 surface expression by diminishing the recycling pool size. Sucrose increases GAT1 surface expression by blocking clathrin- and dynamin-dependent endocytosis, but it does not change the recycling pool size. Protein kinase C decreases surface GAT1 expression by increasing the endocytosis rate, but it does not change the exocytosis rate or the recycling pool size. Based upon estimates of GAT1 molecules in cortical boutons, the present data suggest that approximately 1000 transporters comprise the acutely recycling pool, of which 300 are on the surface in the basal state, and five transporters insert into the plasma membrane every second. This insertion could represent the fusion of one transporter-containing vesicle.

Plasma membrane neurotransmitter transporters determine in part the concentration, time course, and diffusion of extracellular transmitter. Much has been learned about how substrate translocation through the transporter occurs; however, the precise way in which transporter structure maps onto transporter function has not yet been fully elucidated. Here, biochemical and electrophysiological approaches were used to test the hypothesis that intracellular domains of the rat brain GABA transporter (GAT1) contribute to the transport process. Injection of a peptide corresponding to the presumed fourth intracellular loop of the transporter (IL4) into oocytes expressing GAT1 greatly reduced both forward and reverse transport and reduced the transport rate in a dose-dependent manner. Coinjection of the IL4 peptide with a peptide corresponding to the N-terminal cytoplasmic tail of GAT1 reversed the IL4-mediated inhibition; this reversal, and direct binding between these two domains, was prevented by mutagenesis of charged residues in either the IL4 or N-terminal domains. Furthermore, syntaxin 1A, a solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein that inhibits GAT1 transport rates via interactions with the N-terminal tail of GAT1 was unable to regulate the GAT1 IL4 mutant. Together, these data suggest a model in which the GAT1 IL4 domain serves as a barrier for transport, and this barrier can be regulated through intra-molecular and inter-molecular interactions.

A feature of the rat brain gamma-aminobutyric acid transporter GAT1, and other members of the neurotransmitter transporter family, is its regulated redistribution between intracellular locations and the plasma membrane. Recent studies have focused upon defining the signaling molecules that facilitate this redistribution. Agents that promote direct tyrosine phosphorylation of GAT1 promote a relative increase in surface GAT1 levels, and this results from a slowing of the transporter internalization rate. Agents that act to increase protein kinase C (PKC) activity promote a relative decrease in surface GAT1 levels; whether this effect is caused by direct transporter phosphorylation is unknown. The opposing actions of tyrosine kinase activity and PKC activity raise the possibility that the subcellular distribution of GAT1 is associated with mutually exclusive transporter phosphorylation events. The present experiments show that GAT1 is phosphorylated on serine residues in a PKC-dependent manner, but this state is only revealed when GAT1 tyrosine phosphorylation is eliminated or greatly reduced. The relative levels of serine phosphorylation and tyrosine phosphorylation are negatively correlated. The amount of serine phosphorylation is regulated by agents that affect tyrosine phosphorylation, and vice versa. In addition, the ability of agents that affect tyrosine kinase activity to regulate GAT1 serine phosphorylation requires a change in its tyrosine phosphorylation state. These data support the ideas that GAT1 can exist in either of two mutually exclusive phosphorylation states and that the relative abundance of these states determines in part the relative subcellular distribution of the transporter.

Norepinephrine (NE) transporters (NETs) terminate noradrenergic synaptic transmission and represent a major therapeutic target for antidepressant medications. NETs and related transporters are under intrinsic regulation by receptor and kinase-linked pathways, and clarification of these pathways may suggest candidates for the development of novel therapeutic approaches. Syntaxin 1A, a presynaptic solubleN-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET intrinsic activity. NETs colocalize with and bind to syntaxin 1A in both native preparations and heterologous systems. Protein kinase C activation disrupts surface NET/syntaxin 1A interactions and downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A binds the NH2terminal domain of NET, and a deletion of this domain both eliminates NET/syntaxin 1A associations and prevents phorbol ester-triggered NET downregulation. Whereas syntaxin 1A supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two contradictory roles of syntaxin 1A on NET appear to be linked and reveal a dynamic cycle of interactions that allow for the coordinated control between NE release and reuptake.

The loss of functional response upon continuous or repeated exposure to agonist, desensitization, is an intriguing phenomenon if not as yet a well-defined physiological mechanism. However, detailed evaluation of the properties of desensitization, especially for the superfamily of ligand-gated ion channels, reveals how the nervous system could make important use of this process that goes far beyond simply curtailing excessive receptor stimulation and the prevention of excitotoxicity. Here we will review the mechanistic basis of desensitization and discuss how the subunit-dependent properties and regulation of nicotinic acetylcholine receptor (nAChR) desensitization contribute to the functional diversity of these channels. These studies provide the essential framework for understanding how the physiological regulation of desensitization could be a major determinant of synaptic efficacy by controlling, in both the short and long term, the number of functional receptors. This type of mechanism can be extended to explain how the continuous occupation of desensitized receptors during chronic nicotine exposure contributes to drug addiction, and highlights the potential significance of prolonged nAChR desensitization that would also occur as a result of extended acetylcholine lifetime during treatment of Alzheimer's disease with cholinesterase inhibitors. Thus, a clearer picture of the importance of nAChR desensitization in both normal information processing and in various diseased states is beginning to emerge.

Neurotransmitter transporters are regulated through a variety of signal transduction mechanisms which may operate in order to maintain appropriate levels of transmitter in the synaptic cleft. GABA and glycine transporters both interact with components of the neurotransmitter release, such as the SNARE protein syntaxin 1A, suggesting that protein-protein interactions are a common method for regulating members of the neurotransmitter transporter family, and thus, linking the release of transmitter to its subsequent re-uptake. In the present report, the interaction of syntaxin 1A with endogenous serotonin transporters (SERT) expressed in developing thalamocortical neurons is examined. Incubation of thalamocortical cultures with botulinum toxin C1, which specifically cleaves syntaxin 1A, decreased SERT function. Serotonin (5HT) saturation analysis showed that the effect of the toxin was to decrease maximum transport capacity with little change to the affinity of the transporter for 5HT. The 5HT uptake data were consistent with biotinylation experiments showing a decrease in the surface expression of SERT following toxin treatment. In addition, co-immunoprecipitation experiments showed that SERT and syntaxin 1A form a protein complex in these neurons. These data show that components of the transmitter release machinery interact with endogenously expressed amine transporters, and suggest a mechanism for the control of transmitter levels in disorders related to aminergic signaling.

5-HT transporters (SERTs) are transiently expressed in thalamocortical neurons during development, permitting these glutamatergic neurons to co-release 5-HT as a “borrowed” transmitter. The high level of SERT expression in these neurons is likely important in the serotonergic modulation of neocortical circuits and provides a system for examining endogenous SERT regulation. We tested the hypothesis that developmental expression of SERT in thalamocortical neurons is regulated by psychostimulants that are agonists and antagonists of SERT. Cultured thalamocortical neurons from embryonic day 18 rats were examined for SERT expression until P15. In untreated cultures, SERT protein levels peaked at postnatal day 3 (P3) and were absent by P10. Chronic treatment with SERT substrates (5-HT, 3,4-methylenedioxymethamphetamine) increased both peak SERT protein levels (fourfold) and the time course of SERT expression. SERT substrates also shifted the relative functional expression of SERT by redistributing intracellular SERT protein to the plasma membrane. The subcellular redistribution was prevented by PKC activators. SERT antagonists (e.g., fluoxetine, cocaine) reduced total SERT expression levels and the time course of SERT expression. These data (1) show that endogenous SERT is differentially regulated by 5-HT and psychostimulants, (2) indicate that SERT modulation occurs via changes in both total SERT protein levels and subcellular redistribution of the transporter, and (3) suggest that some of the actions of drugs of abuse in neocortical development may be attributable to alterations in SERT expression and concomitant changes in 5-HT signaling.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

Plasma membrane GABA transporters participate in neural signaling through re-uptake of neurotransmitter. The domains of the transporter that mediate GABA translocation and regulate transport are not well understood. In the present experiments, the N-terminal cytoplasmic domain of the GABA transporter GAT1 regulated substrate transport rates. This domain directly interacted with syntaxin 1A, a SNARE protein involved in both neurotransmitter release and modulation of calcium channels and cystic fibrosis transmembrane regulator (CFTR) chloride channels. The interaction resulted in a decrease in transporter transport rates. These data demonstrate that intracellular domains of the GABA and protein-protein interactions regulate substrate translocation, and identify a direct link between the machinery involved in transmitter release and re-uptake.

Recent evidence suggests that some of the syntaxin isoforms may physically interact with and regulate the transport activity of a defined set of membrane transport proteins. This review examines recent studies of the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel which define distinct roles of syntaxin 1A and syntaxin 3 in the regulation of surface expression as well as intrinsic properties of these epithelial ion transporters.

The cystic fibrosis gene encodes a chloride channel, CFTR (cystic fibrosis transmembrane conductance regulator), that regulates salt and water transport across epithelial tissues. Phosphorylation of the cytoplasmic regulatory (R) domain by protein kinase A activates CFTR by an unknown mechanism. The amino-terminal cytoplasmic tail of CFTR was found to control protein kinase A–dependent channel gating through a physical interaction with the R domain. This regulatory activity mapped to a cluster of acidic residues in the NH 2 -terminal tail; mutating these residues proportionately inhibited R domain binding and CFTR channel function. CFTR activity appears to be governed by an interdomain interaction involving the amino-terminal tail, which is a potential target for physiologic and pharmacologic modulators of this ion channel.

Regional-specific differences in nicotinic acetylcholine receptors (nAChRs) were examined using the whole-cell patch clamp technique in rat medial habenula (MHb) slices. The majority of cells in the ventral two thirds of the MHb responded robustly to local pressure application of nAChR agonists. Mean agonist potency profiles in the middle and ventral thirds of the MHb were similar: cytisine was the most potent agonist and DMPP the weakest, consistent with a significant contribution of the beta4 subunit to functional nAChRs in all areas of the MHb. In acutely isolated MHb neurons, the alpha3beta4-selective toxin alpha-CTx-AuIB (1 microM) reversibly blocked approximately 75% of the nicotine-induced currents, as expected for cells solely expressing alpha3beta4 nAChRs. However, the alpha3beta2-selective toxin, alpha-CTx-MII (100 nM), blocked a variable fraction (0-90%) of the MHb nicotinic response implying that beta2 subunits may contribute to some functional receptors. We suggest that the effects of alpha-CTx-MII may arise from interaction with alpha3beta2beta4 subunit-containing nAChRs. This idea is supported by the findings (1) that alpha-CTx-MII antagonizes receptors comprised of alpha3, beta2 and beta4 subunits in Xenopus oocytes, and (2) that a mutant alpha-CTx-MII toxin[H12A], which blocks alpha3beta2beta4 receptors but not alpha3beta2 or alpha3beta4 nAChRs, also reduces nicotinic currents in some MHb neurons. Overall these data imply that most functional nAChRs on MHb cells contain at least alpha3 and beta4 subunits, and that a variable subpopulation additionally contains the beta2 subunit.