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.

Bacteria belonging to the order Actinomycetales produce most microbial metabolites thus far described, several of which have found applications in medicine and agriculture. However, most strains were discovered by their ability to produce a given molecule and are, therefore, poorly characterized physiologically and genetically. Thus, methodologies for genetic manipulation of actinomycetes are not available and efficient tools have been developed for just a few strains. This constitutes a serious limitation to applying molecular genetics approaches to strain development and structural manipulation of microbial metabolites. To overcome this hurdle, we have developed bacterial artificial chromosomes (BAC) that can be shuttled among Escherichia coli, where they replicate autonomously, and a suitable Streptomyces host, where they integrate site-specifically into the chromosome. The existence of gene clusters and of genetically amenable host strains, such as Streptomyces coelicolor or Streptomyces lividans, makes this a sensible approach. We report here that 100 kb segments of actinomycete DNA can be cloned into these vectors and introduced into genetically accessible S. lividans, where they are stably maintained in integrated form in its chromosome.

The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe2+ and manganese, not Fe3+ or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe2+ in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.

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.

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

Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (τ/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and τ/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents.