VGLUT Transports Chloride and Potassium

As discussed in section 3.5.2, the effect of Cl^- on both components of ∆µH+ was larger in glutamatergic compared to GABAergic SVs. Moreover, in the presence of Cl^-, glutamatergic SVs acidified significantly faster than GABAergic SVs. Together, this implies higher Cl^- influx in glutamatergic SVs. Since ClC3 is present on both vesicle subtypes in comparable quantities (Gronborg et al., 2010), it is reasonable to attribute

102| Discussion

the additional Cl^- influx in glutamatergic SVs to VGLUT. This is in line with previous reports where the loss of VGLUT1 (Schenck et al., 2009), but not ClC3 (Riazanski et al., 2011; Schenck et al., 2009), significantly impaired Cl^- induced acidification in brain-purified SVs, whose majority are glutamatergic. This notion has become quite controversial, with some supporting evidence (Bellocchio, 2000; Schenck et al., 2009) and some opposition (Hartinger and Jahn, 1993; Juge et al., 2006). Recently however, more direct evidence for Cl^- conductance of VGLUT was provided by measuring Cl -transport in VGLUT1-reconstituted liposomes using a Cl^--sensitive fluorescent probe (Preobraschenski et al., 2014). In this study, as described in section 1.2.2.3, VGLUT was proposed to possess two anion binding sites, one of which binds to Cl^- and accelerate the conformational switch of the transporter and the second one preferentially binds to glutamate but can also be occupied by Cl^- in the absence of glutamate (Preobraschenski et al., 2014). In this thesis, the measurements were also performed in the absence of glutamate. According to the proposed model, when Cl^- is added to the bath solution, it binds to both the Cl^- binding site and with lower affinity to the glutamate binding site, which the first facilitates the conformational change and the latter transports Cl^- into the lumen. This can explain the observed greater Cl^- influx in glutamatergic SVs in the absence of glutamate.

These results are consistent with the proposed model and demonstrate that the glutamate binding site of the VGLUT can indeed contributes to Cl^- transport as well. This transport of Cl^- but in the reverse direction, i.e. from the lumen to cytoplasm, would significantly facilitate glutamate transport under physiological conditions. At the early phase after endocytosis and during the first transport cycles of glutamate transport, the luminal concentration of Cl^- is high while luminal glutamate is low. Thus, Cl^- can bind to the glutamate binding site when VGLUT is in state II (i.e. the substrate binding pocket is towards the vesicular lumen) (Preobraschenski et al., 2014). This leads to efflux of Cl -from the lumen of early-endocytosed vesicles and allows for the loading of the same concentration of glutamate without any net change in membrane potential, which would substantially enhance glutamate loading. Moreover, it helps to maintain the osmotic balance during vesicle filling. On the other hand, CIC3 can use the proton gradient established by V-ATPase to maintain luminal [Cl^-] and thus allow VGLUT to continue Cl -/glutamate exchange.

However, as discussed above, Cl^- transport via ClC3 into the lumen of SVs shunts ∆ψ, which would dissipate the main driving force for glutamate uptake. Therefore, another

Discussion |103 compensatory mechanism that tips the balance of the electrochemical components back towards ∆ψ would be very beneficial to neurotransmitter filling in glutamatergic SVs.

Indeed, it has been shown that SVs can convert ∆pH to ∆ψ via cation/H⁺ exchange mechanisms (Goh et al., 2011). In order to test whether such an exchange mechanism is particularly employed by glutamatergic SVs, one can compare the effect of Na⁺ and K⁺ on the ∆µH+ in these vesicles with the response of GABAergic SVs, whose reliance on ∆ψ is significantly lower. In fact, as shown in Figure ‎3-17 and Figure ‎3-18, a significantly greater K⁺-induced alkalization was observed in glutamatergic compared to GABAergic SVs, which in contrast to Na⁺-induced alkalinization, was not blocked by the NHE inhibitor EIPA. The greater alkalinization of glutamatergic SVs by K⁺ as well as its resistance to EIPA indicate that VGLUT is responsible for the K⁺ transport into the lumen of SVs. This corroborates with the recently reported K⁺/H⁺ exchange by VGLUT (Preobraschenski et al., 2014). Moreover, consistent with the measured effect of K⁺ on

∆ψ, it was observed that dissipation of the membrane potential upon glutamate uptake was significantly mitigated by the presence of K⁺ (Figure ‎3-24B). This implies that stoichiometry of K⁺/H⁺ exchange by VGLUT is not 1:1. Moreover, it further emphasizes the crucial role of VGLUT mediated K⁺/H⁺ exchange as a charge compensating mechanism, which counteracts the effect of Cl^- on the electrochemical gradient and helps to sustain the driving force required for efficient glutamate loading.

A slight K⁺ effect was also observed in GABAergic SVs. Since it has been proposed that NHEs selectively transport Na⁺ and not K⁺ (Milosavljevic et al., 2014), and also due to the resistance of K⁺-induced alkalinization to the NHE inhibitor EIPA, this effect of K⁺ in GABAergic SVs is probably mediated by VGLUT2 on a subset of these SVs (Zander et al., 2010).

4.6 Na

/H

Exchange Stimulates Vesicle Loading in both Glutamatergic and GABAergic SVs

Na⁺-induced alkalinization was measured to be equal in glutamatergic and GABAergic SVs (Figure ‎3-18), indicating that a common protein is mediating Na⁺ influx. Since the effect of Na⁺ was blocked by EIPA, an inhibitor of most NHE isoforms (Goh et al., 2011), and also a quantitative proteomics study revealed equal expression of NHEs on both vesicle subclasses (Gronborg et al., 2010), this Na⁺ influx must be caused by NHEs, most probably NHE6 which resides on SVs (Preobraschenski et al., 2014). In case of

104| Discussion

glutamatergic SVs, which mainly rely on ∆ψ for neurotransmitter loading, Na⁺/H⁺ exchange by NHE6 in cooperation with K⁺/H⁺ exchange by VGLUT stimulates neurotransmitter loading by maintaining ∆ψ at the expense of ∆pH. In addition, in GABAergic SVs, NHE6 counteracts the effect of Cl^- on ∆µH+ and together with ClC3 will provide a balance between ∆pH and ∆ψ, both of which are required by VGAT for efficient GABA loading.

Moreover, it has been recently proposed that NHE7, whose primary structure resembles that of NHE6 on SVs (Nakamura et al., 2005), can operate in a reverse mode, exchanging luminal Na⁺ for cytosolic H⁺ (Milosavljevic et al., 2014). This suggests that NHE6, in addition to its role as a regulator of ∆µH+, might also contribute to Na⁺ efflux from the lumen of early endocytosed SVs, which contain more than 100 mM NaCl in both glutamatergic and GABAergic SVs.