Matthew L. Beckman

Transport rates of GABA transporters: regulation by the N-terminal domain and syntaxin 1A.

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.

Neurotransmitter Transporters: Regulators of Function and Functional Regulation

Reliable chemical neurotransmission requires spatial and temporal control of neurotransmitter in the synaptic cleft. Neurotransmitter transporters, located on neurons and glia at or near the synapse, are key participants in this process. Historically, neurotransmitter transporters have been thought to participate by removing transmitter from the synaptic cleft; exciting new data reveal that this is only one route by which transporters regulate synaptic events. In addition, recent evidence suggests that transporters and transporter function can be regulated through multiple mechanisms. Taken together, these findings are the impetus for the present review, the title of which is chosen to denote the dynamic interplay among the factors that control neurotransmitter transporter function and the control of synaptic signaling by neurotransmitter transporters. Herein, we review (i) the ways in which neurotransmitter transporters are regulators of neuronal function and (ii) the ways by which the transporters themselves are regulated. We apologize at the outset for excluding a number of excellent scientific contributions from many different laboratories. We have deferred discussion of related topics including vesicular transporters, sodium-dependent glucose transporters, transporter classification, and transporter structure/function; other recent review articles cover these topics in detail. Transporters Regulate Synaptic Signaling

Desensitization of Nicotinic Receptors in the Central Nervous System

hronic exposure to nicotine renders some CNS neuronal nicotinic acetylcholine receptors (nAChRs) “permanently” desensitized. These long-lived inactive receptor states are likely to underlie the development of tolerance to nicotine and may be responsible for some of the more long-term addictive properties of this drug. An understanding of nicotine dependency would benefit from knowledge of the neuronal factors that control desensitization of nAChRs. During recordings of nAChR channel activity in excised outside-out patches from medial habenula (MHb) neurons, there is a time-dependent increase in the rate of onset of receptor desensitization, assessed from the decay of macroscopic currents produced by brief pulses of nicotine. This alteration in desensitization precedes a complete rundown in channel activity. FIGURE 1A shows that after rundown of channel activity in an outsideout patch, a partial recovery of channel function is possible, provided that nAChRs are not exposed to nicotine for a prolonged period. These data are consistent with the idea that rundown of channel activity results from a build-up of nAChRs in a nonfunctional desensitized state; this may occur because recovery from desensitization is slowed due to the loss of certain factors upon patch formation. Knowledge of the factors that control how nAChRs exit from the desensitized states of the receptor should lead to an understanding of how neurons control the level of nAChR activity. Because receptors at synapses are only likely to be exposed to neurotransmitter very transiently, the physiological importance of these slowly reached desensitized states is unknown. However, because desensitized receptor conformations have high affinities for agonist, these states will become occupied during the continuous presence of drug, for example, as occurs during smoking. Furthermore, because certain nAChRs appear to become “permanently” inactivated during chronic exposure to nicotine, we hypothesize that nAChRs become trapped in a desensitized state(s) due to a long-term biochemical alteration. Chronic nicotine, in addition to causing a long-term inactivation of nAChRs, causes an up-regulation in the number of high-affinity [H]nicotine (α4β2 nAChR subunit-containing) binding sites. In accordance with these observations, we have shown that tobacco-related levels of nicotine (≈30–300 nM) will preferentially interact with the desensitized state of α4containing nAChRs: in Xenopus oocytes expressing rat α4β2 nAChRs, nicotine opens channels with an EC50 of ≈10 μM, whereas the IC50 for inducing receptor desensitization is ≈60 nM. After a 30 min exposure to 300 nM nicotine, receptors recover from desensitization with a relatively slow time course. Consistent with the idea that nAChRs could become trapped in the desensitized state, the rate of recovery from desensitization is strongly influenced by

Substrates and temperature differentiate ion flux from serotonin flux in a serotonin transporter

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.

PICKing on transporters

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.

The Ups and Downs of Neurotransmitter Transporters

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.

Expression of functional scorpion neurotoxin Lqq-V in E.coli.

We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.