4. Permanent depolarization to -40 mV appears to immobilize the slowly moving charge. Depolarization to -20 mV immobilizes both charge movements, and uncovers the presence of a third charge which seems to correspond to Charge 2 (cf.

Cloning of ion-coupled transporters and their heterologous expression has allowed insights on the molecular mechanism of translocation (Hediger et al. 1987;Lester et al. 1994). Among the best-studied systems so far are those involved in the reuptake of amino acid neurotransmitters, such as the GAT1 transporter (Mager et al. 1993) or the 5-hydroxytryptamine (5-HT) transporter (Mager et al. 1994). Electrophysiological investigations on these molecules have revealed interesting aspects of the ion and substrate translocating steps. The permeation properties of the neurotransmitter transporters have been studied in detail and an 'alternating-access' mechanism of transport has been envisaged (Lester et al. 1994). In this view the transporter resembles a channel with gates at both ends, which open and close, exposing the internal lumen to either the extra-or intracellular sides. More recently, however, a different scheme based on the multi-ion, single-file model developed for channels (Hille, 1992) has been proposed (Su et al. 1996).In this kind of model the transporter acts as a 'sticky' channel in which substrates bind to specific sites during the permeation process. The advantages of this model are that it accounts for the uncoupled currents and for the variable stoichiometry observed in some transporters (Cammack et al. 1994;Mager et al. 1994;Risso et al. 1996). Another interesting feature exhibited by neurotransmitter transporters expressed at high density in Xenopus oocytes is the existence of so-called 'pre-steady-state currents' induced by step changes in the membrane voltage (Loo et al. 1993; Mager et al. 1993Mager et al. , 1996. These pre-steady-state currents are similar to the better-known 'gating currents' of voltage-dependent channels; these are believed to arise from

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The effect of the mutation K448E in the rat GABA transporter rGAT1 was studied using heterologous expression in Xenopus oocytes and voltage clamp. 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 μm at −60 mV). 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. 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. 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. 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.

Complementary RNA, derived from the intestine of the sea bass Dicentrarchus labrax and putatively coding for a pH-dependent oligopeptide transporter PepT1 (SLC15 family), was injected in Xenopus oocytes that were subsequently tested with electrophysiological techniques. Transport-associated currents were observed when various di- or tripeptides were applied at concentrations ranging between 0.1 and 10 mM. No currents were generated by histidine nor by other single amino acids. Sea bass PepT1 also exhibited presteady-state currents in the absence of substrates. Acidic pH slowed down the relaxation time constant of these currents and shifted both Q/V and tau/V relationships toward more positive voltages. Michaelis-Menten analysis of the transport currents showed an increase in apparent substrate affinity at acidic pH, which was very similar to that exhibited by the related transporter from zebrafish (Danio rerio), but in contrast, did not demonstrate a significant effect of pH on the maximal transport current.

In Novikoff hepatoma cell pairs studied by double perforated patch clamp (DPPC), brief (20 s) exposure to 20 microM arachidonic acid (AA) induced a rapid and reversible uncoupling. In pairs studied by double whole-cell clamp (DWCC), uncoupling was completely prevented by effective buffering of Cai2+ with BAPTA. Similarly, AA (20 s) had no effect on coupling in cells perfused with solutions containing no added Ca2+ (SES-no-Ca) and studied by DPPC, suggesting that Ca2+ influx plays an important role. Parallel experiments monitoring [Ca2+]i with fura-2 showed that [Ca2+]i increases with AA to 0.7-1.5 microM in normal [Ca2+]o, and to approximately 400 nM in SES-no-Ca solutions. The rate of [Ca2+]i increase matched that of Gj decrease, but [Ca2+]i recovery was faster. In cells studied by DWCC with 2 mM BAPTA in the pipette solution and superfused with SES-no-Ca, long exposure (1 min) to 20 microM AA caused a slow and virtually irreversible uncoupling. This result suggests that AA has a dual mechanism of uncoupling: one dominant, fast, reversible, and Ca(2+)-dependent, the other slow, poorly reversible, and Ca(2+)-independent. In contrast, uncoupling by oleic acid (OA) or halothane was insensitive to internal buffering with BAPTA, suggesting a Ca(2+)-independent mechanism only.

The effects of temperature on the Q Q-aminobutyric acid (GABA) uptake and on the presteady-state and transportassociated currents of the GABA cotransporter, rat Q Q-aminobutyric acid transporter 1 (rGAT1), have been studied using heterologous oocyte expression and voltage-clamp. Increasing temperature from 15 to 30³C 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 d d versus V curve was also negatively shifted by increasing temperatures. Analysis of the outward (K K) and inward (L L) rate constants as functions of temperature showed that they are affected differently, with a Q 10 = 3.4 for K K and Q 10 = 1.5 for L L. 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. ß

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