Characterisation of glutamate transport by DVGLUT, eat-4 and VGLUT2

To characterize the transport of the VGLUT1 homologues some key parameters were chosen since it was not feasible to express all proteins in the same amounts that would be

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necessary to characterize the transporters with the same depth as VGLUT1. First, it was important to see if the dependence on the internal anion is preserved among the isoforms.

Second, the activation by extravesicular Cl^- was analyzed by measuring the uptake in the presence of low millimolar concentrations of Cl^- or in the absence of Cl^-. These two conditions were also measured in the presence of Nigericin. In this way some major conclusions about the energy dependence could be drawn from relatively few experiments, hence saving protein samples. DVGLUT was characterised more detailed by measuring uptake also in the presence of various external Cl^- concentrations (see Fig 3.25) because the neuromuscular junction of Drosophila is a widely used synapse in electrophysiological studies and therefore more amenable to extensive investigations. For this common model organism more information on the glutamate loading mechanism of SVs would be most beneficial.

3.9.1 DVGLUT transports glutamate but no aspartate

In a first approach the specificity and ATP-dependence of the glutamate uptake by reconstituted DVGLUT was tested along with the internal anion. The uptake was measured under standard conditions (see 2.7) with 4 mM extravesicular Cl^- for Cl^--and gluconate-loaded liposomes and compared to the transport in the absence of ATP and/or the presence of 10 mM aspartate. Aspartate was not recognized as a substrate,

showing that the specificity for glutamate is preserved also in this isoform like for all mammalian VGLUTs (Bellocchio et al., 2000; Fremeau et al., 2002; Fremeau et al., 2001) (Fig. 3.34a). The uptake appeared to be only slightly influenced by the intravesicular anion under these conditions and was strictly dependend on the activity of the proton pump. The glutamate uptake of Cl^- loaded DVGLUT-liposomes was lower in the absence of ATP

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compared to Cl^- loaded VGLUT1 liposomes were some uptake occurred (Fig. 3.34b and see also Fig. 3.23b). However, the signal is relatively low, making it difficult to draw conclusions with certainty.

3.9.2 DVGLUT shows a biphasic dependence on extravesicular Cl^- and only minimal enhancement by intravesicular Cl

-To test if the biphasic dependence, characteristic for mammalian VGLUTs is preserved in DVGLUT, a hitherto unknown parameter, the uptake of glutamate was tested under different extravesicular Cl^- concentrations (Fig. 3.35). It is evident that the biphasic response is also present in DVGLUT, such that low millimolar Cl^- concentrations enhance the glutamate translocation. Interestingly, however, the uptake is not strongly enhanced by intravesicular Cl^-. This is most prominent in the absence of extravesicular Cl^- and stands in strong contrast to VGLUT1 (Fig. 3.35 red and golden traces).

3.9.3 The glutamate translocation by DVGLUT and eat-4 depends largely on pH

To gain some insight into the energy dependence of the evertebrate iosoforms, DVGLUT and eat-4 were reconstituted along with VGLUT2 and VGLUT1 and uptake was measured in the presence or absence of Nigericin at 0 mM and 4 mM external Cl^- respectively. This was done for both gluconate- and Cl^--loaded liposomes (Fig. 3.36). The data shows that the glutamate transport by DVGLUT is only slightly enhanced in the presence of luminal Cl^- ions when external Cl^- is missing. Furthermore, the transport is driven mainly by pH, since Nigericin almost abolishes the uptake in the presence of extravesicular Cl -independent of the internal anion. Thus DVGLUT apparently is not capable to operate as a Cl^-/glutamate exchanger but instead transports glutamate most likely by exchange with protons. The pH driven component of glutamate uptake is also present in VGLUT1 and overlaps with the -driven antiport when luminal Cl^- ions are present. The reduction in the transport of glutamate by nigericin application is therefore not as strong as for DVGLUT. The difference in transport by DVGLUT is most obvious in the absence of external Cl^- where mammalian VGLUTs transport efficiently when antiport with Cl^- is possible. Eat-4 shows no enhancement by luminal Cl^- and apparently is even inhibited by high luminal Cl^- concentrations. This is a very surprising finding and currently difficult to explain. Eat-4 also shows the most pronounced dependence on pH as Nigericin has the strongest effect - almost abolishing the transport. The C. elegans homologue is like other VGLUTs activated by low millimolar extravesicular Cl^- concentrations and, judging from the effect of nigericin, by establishment of a pH in the lumen. However, further investigations are needed to get a clearer picture of glutamate translocation by eat-4. For instance, it could be that even higher extravesicular Cl^- concentrations enhance the glutamate uptake further.

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3.9.4 Murine VGLUT2 shows the same energy dependence pattern as VGLUT1 resembles largely the one observed for VGLUT1 and indicates that mammalian VGLUTs are different in their transport characteristics from invertebrate VGLUTs. Since the synapses where VGLUTs operate are serving different functions in invertebrates and vertebrates, neuromuscular junctions and CNS respectively, the different demands in the specific physiological function of those synapses may reflect the diversity in transport mechanisms. Again, it is not possible yet to conclude certainly from the few data available, however, the observations are relatively clear and could help to understand the details of the glutamate translocation in these isoforms.

3.9.5 DVGLUT exhibits a Cl^- shunting activity in liposome acidification

Interestingly, DVGLUT is still enhanced by extravesicular Cl^- in a very similar manner as the mammalian VGLUTs, although intravesicular Cl^- does not seem to have an effect on the transport. This immediately raises the question whether Cl^- can permeate through DVGLUT and could help to understand if the influx and efflux of Cl^- in VGLUT1 are mediated by the same mechanism. Therefore DVGLUT was assayed by AO quenching in the presence of 100 mM extravesicular Cl^- (Fig. 3.37). Indeed, Cl^- permeates DVGLUT, which is compatible to the effect of nigericin that reduces the glutamate uptake when 4 mM external Cl^- is present. The data thus suggests that the entry of Cl^- through VGLUTs is preserved and that the pathway or leak for this entry is different from the efflux that was observed in the mammalian VGLUTs. Since extravesicular Cl^- diminishes the uptake in the presence of nigericin also at low millimolar concentrations in VGLUT1 (see fig. 3.25, red dashed traces) the site for Cl^- entry could indeed be the glutamate binding site. It is conceivable that Cl^- leaks through the glutamate entry site which

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would also explain the competitive character of Cl^-. However it is not possible to fix such a view yet since no mutational or structural information could support this view. It is nevertheless an interesting finding that can help to elucidate the transport mechanism of glutamate and the anion shunting in SVs in future investigations.

3.10 The differences of invertebrate and vertebrate VGLUTs are useful for