The norepinephrine (NE) transporter (NET) mediates the removal of NE from synaptic spaces and is a major target for antidepressants, amphetamine and cocaine. Previously, we have shown that syntaxin 1A (SYN 1A) supports human NET (hNET) cell surface expression, that hNET/SYN 1A interactions are direct and mediated by the hNET N-terminus, and that the hNET/SYN 1A association limits substrate-induced hNET-associated currents [Sung, U., Apparsundaram, S., Galli, A., Kahlig, K.M., Savchenko, V., Schroeter, S., Quick, M.W., Blakely, R.D., 2003. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J. Neurosci. 23, 1697–1709]. These data raise the possibility that the hNET N-terminus, and potentially its interaction with SYN 1A, might regulate other hNET conductance states, including the hNET-mediated leak current. Importantly for monoamine transporters, the leak conductance has been shown to play a critical role in regulating cell membrane potential and possibly neuronal excitability [Quick, M.W., 2003. Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537–549]. Here we demonstrate that deletion of the binding domain for SYN 1A in the NET N-terminus robustly enhances the NET-mediated leak current as well as its selectivity for Cl− permeation under particular intracellular ionic compositions. In addition, we show that the NET N-terminus coordinates the ability of intracellular Na+ and Cl− to regulate the leak conductance. These data suggest that the NET N-terminus regulates and defines the ionic specificity of the NET-mediated leak current.

Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity/ plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na+/Cl--dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (i) a slow process consistent with an exchange mechanism and (ii) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA; when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.

The effects of reducing external Cl– on the electrophysiological properties of the Na+/Cl–-dependent GABA transporter rGAT1 expressed in Xenopus oocytes were investigated. In agreement with a recently proposed kinetic scheme, the effects of Cl– are complex but preserve the mutual relationship that links the transport-associated current, Itr, measured in saturating GABA concentration, and the transient current, Ipre, recorded in the absence of GABA following a voltage step from the holding potential Vh to V. In particular, Itr (V) – Itr (Vh) = r ∫ Ipre (V) dt, where r is the relaxation rate of Ipre at the same membrane potential and Cl– concentration. The model also predicts a relationship between charge relaxation rate and apparent affinity for GABA, which is also verified in the presence of lowered Na+ or Cl– concentrations. In these conditions, the binding rate of GABA to the transporter is increased. All these effects are consistent with the hypothesis that interaction of the organic substrate with rGAT1 induces a conversion from a capacitive to a conductive mode of operation without strongly altering either the amount or the rate of charge movement.

Most cotransporters characteristically display two main kinds of electrical activity: in the absence of organic substrate, transient presteady-state currents (I(pre)) are generated by charge relocation during voltage steps; in the presence of substrate, sustained, transport-associated currents (I(tr)) are recorded. Quantitative comparison of these two currents, in Xenopus oocytes expressing the neural GABA cotransporter rGAT1, revealed several unforeseen consistencies between I(pre) and I(tr), in terms of magnitude and kinetic parameters. The decay rate constant (r) of I(pre) and the quantity of charge displaced to an inner position in the transporter (Q(in)(0)) depended on voltage and ionic conditions. Saturating GABA concentrations, applied under the same conditions, suppressed I(pre) (i.e. Q(in)( infinity ) = 0) and produced a transport-associated current with amplitude I(tr) = Q(in)(0)r. At non-saturating levels of GABA, changes of I(tr) were compensated by corresponding variations in Q(in), such that I(pre) and I(tr) complemented each other, according to the relation: I(tr) = (Q(in)(0) - Q(in)) r. Complementarity of magnitude, superimposable kinetic properties and equal dependence on voltage and [Na(+)](o) point to the uniqueness of the charge carrier for both processes, suggesting that transport and charge migration arise from the same molecular mechanism. The observed experimental relations were correctly predicted by a simple three-state kinetic model, in which GABA binding takes place after charge binding and inward migration have occurred. The model also predicts the observed voltage dependence of the apparent affinity of the transporter for GABA, and suggests a voltage-independent GABA binding rate with a value around 0.64 microM(-1) s(-1).

The effects of temperature on the gamma-aminobutyric acid (GABA) uptake and on the presteady-state and transport-associated currents of the GABA cotransporter, rat gamma-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30 degrees C increased GABA uptake, diminished the maximal value of the relaxation time constant of the presteady-state currents and increased the amplitude of the current associated with the transport of GABA. The curve of the presteady-state charge versus voltage was shifted toward negative potentials by increasing the temperature, while the maximal amount of charge (Q(max)) remained constant; the tau versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (alpha) and inward (beta) rate constants as functions of temperature showed that they are affected differently, with a Q(10)=3.4 for alpha and Q(10)=1.5 for beta. The different temperature coefficients of the rate constants account for the observed shifts. These observations are consistent with a charge moving mechanism based on a conformational change of the protein; the weaker temperature sensitivity of the inward rate constant suggests a rate-limiting diffusional component on this process.

The effects of sodium and chloride on the properties of the sodium‐dependent component of the ‘pre‐steady‐state’ currents of rGAT1, a GABA cotransporter of the Na+‐Cl−‐dependent family, were studied using heterologous oocyte expression and voltage clamp. Reductions in either extracellular sodium or chloride shifted the charge‐voltage (Q‐V) and time constant‐voltage (τ‐V) characteristics of the process towards more negative potentials. The shift induced by sodium (TMA+, tetramethylammonium substitution) was stronger than that induced by chloride (acetate substitution), and the shift of τ was accompanied by a decrease in its maximum value. Increasing extracellular Ca2+ did not produce significant shifts in Q‐V and τ‐V curves. The negative shift of the Q‐V curve upon chloride reduction and the decrease in the value of the relaxation time constant, τ, when either sodium or chloride were lowered, contrasted with the prediction of the Hill‐Boltzmann interpretation of the process. Analysis of the unidirectional rate constants under different conditions revealed that both sodium and chloride shifted the outward rate more than the inward rate; furthermore, the shifts induced by sodium were larger than those induced by chloride. These observations are qualitatively compatible with the existence of a selective vestibule at the mouth of the transporters, acting similarly to a Donnan system.

1. The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 2. At neutral pH, the transport-associated current vs. voltage (I-V) relationship of the mutated transporter was different from wild-type, and the pre-steady-state currents were shifted towards more positive potentials. The mutated transporter showed an increased apparent affinity for Na+ (e.g. 62 vs. 152 mM at -60 mV), while the opposite was true for GABA (e.g. 20 vs. 13 microM at -60 mV). 3. In both isoforms changes in [Na+]o shifted the voltage dependence of the pre-steady-state and of the transport-associated currents by similar amounts. 4. In the K448E form, the moved charge and the relaxation time constant were shifted by increasing pH towards positive potentials. The transport-associated current of the mutated transporter was strongly reduced by alkalinization, while acidification slightly decreased and distorted the shape of the I-V curve. Accordingly, uptake of [3H]GABA was strongly reduced in K448E at pH 9.0. The GABA apparent affinity of the mutated transporter was reduced by alkalinization, while acidification had the opposite result. 5. These observations suggest that protonation of negatively charged residues may regulate the Na+ concentration in the proximity of the transporter. Calculation of the unidirectional rate constants for charge movement shows that, in the K448E form, the inward rate constant is increased at alkaline pH, while the outward rate constant does not change, in agreement with an effect due to mass action law. 6. A possible explanation for the complex effect of pH on the transport-associated current may be found by combining changes in local [Na+]o with a direct action of pH on GABA concentration or affinity. Our results support the idea that the extracellular loop 5 may participate to form a vestibule to which sodium ions must have access before proceeding to the steps involving charge movement.