Accumulating genetic evidence indicates that the primate-specific gene locus G72/G30 is related to schizophrenia: it encodes for the protein pLG72, whose function is still the subject of controversy. We recently demonstrated that pLG72 negatively affects the activity of human d-amino acid oxidase (hDAAO, also related to schizophrenia susceptibility), which in neurons and (predominantly) in glia is expected to catabolize the neuromodulator d-serine. The d-serine regulation mechanism relying on hDAAO-pLG72 interaction does not match with the subcellular localizations proposed for hDAAO (peroxisomes) and pLG72 (mitochondria). By using glioblastoma U87 cells transfected with plasmids encoding for hDAAO and/or pLG72 we provide convergent lines of evidence that newly synthesized hDAAO, transitorily present in cytosol before being delivered to the peroxisomes, colocalizes and interacts with pLG72 which we propose to be exposed on the external membrane of mitochondria. We also report that newly synthesized cytosolic hDAAO is catalytically active, and therefore pLG72 binding-and ensuing hDAAO inactivation-plays a protective role against d-serine depletion.

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.

A filamentous cyanobacterium, belonging to the Order of Oscillatoriales, was found to be responsible for a toxic algal bloom in Lake Varese, Italy, during the summer of 1997. Morphological characters, as well as near complete 16S rRNA gene sequencing, revealed that the dominant species of the bloom was most closely related to the genus Planktothrix. In addition, genetic analysis of the phycocyanin operon of Planktothrix sp. FP1 revealed a novel primary structure, previously undescribed within the cyanobacteria, which was used as a genetic marker for rapid detection and identification of this toxic strain. The occurrence of saxitoxin (STX), a principal toxin in paralytic shellfish poisoning (PSP), was confirmed in the natural bloom sample by both pre-column and post-column derivatization high-performance liquid chromatography (HPLC) analyses, and eventually by liquid chromatography/mass spectrometry (LC/MS). The toxicity of this field sample was also revealed by electrophysiological assays in which the extract inhibited 90% of the voltage-dependent Na current in human neuroblastoma cells at the STX concentration of 80 nM. The cultured strain showed a lower physiologic activity than the bloom sample (67% blockage of Na current at a toxin concentration of 200 nM), and STX was detected only by pre-column HPLC, indicating the presence of a compound structurally close to STX. Chemical and molecular genetic analyses performed here add Planktothrix sp. FP1 to the growing list of diverse cyanobacterial species capable of synthesizing STX and its related compounds.

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

The substrate specificity of KAAT1, a Na + -and K + -dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, constructed on the basis of the atomic structure of a leucine-transporting bacterial member of the family, which indicates the participation of this residue in the leucine-binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K + . These results may be interpreted by a kinetic scheme in which, in presence of Na + , the leucine-bound state of the transporter is relatively stable, while in presence of K + and at negative potentials the progression of the leucine-bound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity.