Transient Currents in the Glycine Cotransporter GlyT1 Reveal Different Steps in Transport Mechanism.

The relation between presteady-state (transient) currents elicited by voltage steps in the absence of organic substrate and transport-associated currents in the presence of glycine was investigated in Xenopus oocytes expressing the neuronal glycine transporter GlyT1b. Saturating amounts of glycine converted the transient currents in steady transport currents. Analysis of the transient currents abolished by the substrate confirmed the intramembrane nature of the underlying charge movement process. The sigmoidal Q/V relationship had a moderate slope consistent with the known GlyT1b stoichiometry. The transient currents were best fitted by the sum of two exponentials, with the slow time constant (tau (slow)) being in the order of tens of milliseconds. The apparent affinity for glycine was in the micromolar range and voltage-dependent, slightly decreasing at positive potentials. Numerical simulations show that a simplified, three-state model is sufficient to explain the main features of GlyT1b operation

OLIGOMERIC STRUCTURE OF THE NEUTRAL AMINO ACID TRANSPORTERS KAAT1 AND CAATCH1

The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the Na(+)/Cl(-)-dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the NH(2) or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeuT(Aa) atomic structure and confirm that the COOH termini of the adjacent subunits are closer than NH(2) termini

GABA transporter lysine 448: a key residue for tricyclic antidepressants interaction.

The effects of three tricyclic antidepressants (TCAs) and two serotonin selective reuptake inhibitors (SSRIs) have been studied with an electrophysiological approach on Xenopus laevis oocytes expressing the rat GABA (gamma-Aminobutyric-acid) transporter rGAT1. All tested TCAs and SSRIs inhibit the GABA-associated current in a dose-dependent way with low but comparable efficacy. The pre-steady-state and uncoupled currents appear substantially unaffected. The efficacy of desipramine, but not of the other drugs, is strongly increased in the lysine-glutamate or -aspartate mutants K448E and K448D. Comparison of I(max) and K(0.5GABA) in the absence and presence of desipramine showed that both parameters are reduced by the drug in the wild-type and in the K448E mutant. This suggests an uncompetitive inhibition, in which the drug can bind only after the substrate, an explanation in agreement with the lack of effects on the pre-steady-state and leak currents, and with the known structural data