Sela Mager

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

Conducting states of a mammalian serotonin transporter

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Listening to Neurotransmitter Transporters

Neurotransmitter transporters thus have properties not imagined just a few years ago. Do any of these properties explain the puzzle posed at the beginning: a ubiquitously expressed molecule forms the target for a class of drugs that produce highly specific behavioral effects? In the opinion of most investigators this selective therapeutic action arises from processes, such as gene activation and synaptic remodeling, that occur during the two or three weeks of treatment required before fluoexetine and similar drugs produce their behavioral effects. It remains possible that the therapeutic processes depend on cell-specific variations in the physiological properties of neurotransmitter transporters reviewed here, or that changes in these physiological properties account at least partially for the therapeutic effect. We suspect, however, that the chief benefit of listening more carefully to neurotransmitter transporters will be an increased appreciation for the synapse as an electrochemical machine, specialized to function on a time scale of milliseconds and a distance scale of micrometers.

A multi-substrate single-file model for ion-coupled transporters

Ion-coupled transporters are simulated by a model that differs from contemporary alternating-access schemes. Beginning with concepts derived from multi-ion pores, the model assumes that substrates (both inorganic ions and small organic molecules) hop a) between the solutions and binding sites and b) between binding sites within a single-file pore. No two substrates can simultaneously occupy the same site. Rate constants for hopping can be increased both a) when substrates in two sites attract each other into a vacant site between them and b) when substrates in adjacent sites repel each other. Hopping rate constants for charged substrates are also modified by the membrane field. For a three-site model, simulated annealing yields parameters to fit steady-state measurements of flux coupling, transport-associated currents, and charge movements for the GABA transporter GAT1. The model then accounts for some GAT1 kinetic data as well. The model also yields parameters that describe the available data for the rat 5-HT transporter and for the rabbit Na(+)-glucose transporter. The simulations show that coupled fluxes and other aspects of ion transport can be explained by a model that includes local substrate-substrate interactions but no explicit global conformational changes.

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation.

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.

Glutamate‐101 is critical for the function of the sodium and chloride‐coupled GABA transporter GAT‐1

We have investigated the possible role of selected negatively‐charged amino acids of the sodium and chloride‐coupled GABA transporter GAT‐1 on sodium binding. These residues located adjacent to putative transmembrane domains and which are conserved throughout the large superfamily of neurotransmitter transporters were changed by site‐directed mutagenesis. The functional consequences were that one of the residues, glutamate‐101, was critical for transport. Its replacement by aspartate left only 1% of the activity, and no activity could be detected when it was replaced by other residues. Expression levels and targeting to the plasma membrane of the mutant transporters appeared normal. Transient sodium currents were not observed in the mutants, and increased sodium concentrations did not affect the percentage of wild type transport of the E101D mutant. It is concluded that residue glutamate‐101 is critical for one or more of the conformational changes of GAT‐1 during its transport cycle.

MEASUREMENT OF TRANSIENT CURRENTS FROM NEUROTRANSMITTER TRANSPORTERS EXPRESSED IN XENOPUS OOCYTES

Electrophysiological measurements add new dimensions to the study of neurotransmitter transporters. (1) One can perform measurements with high temporal resolution (however, uptake of radioactive substrate is limited in that it cannot resolve events that occur within 1 sec, which is greater than the time of a single transport cycle). (2) Electrophysiology provides information about partial steps in transport cycles, including the fact that ion binding and dissociation at transporters can generate currents, which provides new insights about ion-transporter interaction. (3) Electrophysiology provides information about single transporter molecules, from patch-clamp recordings of single-channel activity of neurotransmitter transporters. At present, little is known about the molecular mechanisms that underlie transport. Electrophysiological measurements of ion binding and permeation contribute to the analysis of mutations that affect transport. Electrophysiology may help to identify amino acids and domains in neurotransmitter transporters that participate in specific ways in the transport process, such as ion neurotransmitter binding, permeation pathways, voltage sensors, and gates. In combination with spectroscopic measurements, it may also be possible to identify the actual conformational changes of the proteins that enable substrate translocation.

Topological localization of cysteine 74 in the GABA transporter, GAT1, and its importance in ion binding and permeation

Xenopus oocytes expressing the GABA transporter GAT1 were exposed to membrane‐impermeant sulfhydryl reagents, resulting in decreased GABA transport current, decreased capacitive charge movements, and increased Na+ and Li+ leakage currents. Mutation of cysteine 74 to alanine (C74A) eliminated these effects. The W68S and W68L mutations significantly increased and decreased the transporters sensitivity, respectively, to sulfhydryl reagents. At each of the positions 73 through 76, cysteine residues were accessible to external MTSET. These findings, together with recent evidence placing the HD2–HD3 loop on the extracellular side, suggest that the HD2 region does not traverse the membrane.