Publications

• Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism.

  Eriksen J., Chang R., McGregor M., Silm K., Suzuki T., Edwards R.H.

  The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restr ... >> More

  The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restricts flux to acidic membranes such as synaptic vesicles. However, vesicular transport of the principal excitatory transmitter glutamate depends primarily on membrane potential, which would drive non-vesicular efflux, and the role of protons is unclear. Adapting electrophysiology to record currents associated with the vesicular glutamate transporters (VGLUTs), we characterize a chloride conductance that is gated by lumenal protons and chloride and supports glutamate uptake. Rather than coupling stoichiometrically to glutamate flux, lumenal protons and chloride allosterically activate vesicular glutamate transport. Gating by protons serves to inhibit what would otherwise be substantial non-vesicular glutamate efflux at the plasma membrane, thereby restricting VGLUT activity to synaptic vesicles. << Less

  Neuron 90:768-780(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Selective transport of neurotransmitters and modulators by distinct volume-regulated LRRC8 anion channels.

  Lutter D., Ullrich F., Lueck J.C., Kempa S., Jentsch T.J.

  In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and it ... >> More

  In response to swelling, mammalian cells release chloride and organic osmolytes through volume-regulated anion channels (VRACs). VRACs are heteromers of LRRC8A and other LRRC8 isoforms (LRRC8B to LRRC8E), which are co-expressed in HEK293 and most other cells. The spectrum of VRAC substrates and its dependence on particular LRRC8 isoforms remains largely unknown. We show that, besides the osmolytes taurine and <i>myo</i>-inositol, LRRC8 channels transport the neurotransmitters glutamate, aspartate and γ-aminobutyric acid (GABA) and the co-activator D-serine. HEK293 cells engineered to express defined subsets of LRRC8 isoforms were used to elucidate the subunit-dependence of transport. Whereas LRRC8D was crucial for the translocation of overall neutral compounds like <i>myo</i>-inositol, taurine and GABA, and sustained the transport of positively charged lysine, flux of negatively charged aspartate was equally well supported by LRRC8E. Disruption of LRRC8B or LRRC8C failed to decrease the transport rates of all investigated substrates, but their inclusion into LRRC8 heteromers influenced the substrate preference of VRAC. This suggested that individual VRACs can contain three or more different LRRC8 subunits, a conclusion confirmed by sequential co-immunoprecipitations. Our work suggests a composition-dependent role of VRACs in extracellular signal transduction. << Less

  J. Cell Sci. 130:1122-1133(2017) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter.

  Preobraschenski J., Cheret C., Ganzella M., Zander J.F., Richter K., Schenck S., Jahn R., Ahnert-Hilger G.

  Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (P_i). It is still unclear how VGL ... >> More

  Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (P_i). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH⁺ with inversely directed P_i transport coupled to the Na⁺ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and P_i using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH⁺-dependent fashion, with glutamate preferred over P_i. When facing the extracellular space, P_i is transported in a Na⁺-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and P_i homeostasis within glutamatergic neurons. << Less

  Cell Rep. 23:535-545(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Functional characterization of vesicular excitatory amino acid transport by human sialin.

  Miyaji T., Omote H., Moriyama Y.

  Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not tr ... >> More

  Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions. << Less

  J. Neurochem. 119:1-5(2011) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Molecular composition and heterogeneity of the LRRC8-containing swelling-activated osmolyte channels in primary rat astrocytes.

  Schober A.L., Wilson C.S., Mongin A.A.

  <h4>Key points</h4>The volume-regulated anion channel (VRAC) is a swelling-activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as ... >> More

  <h4>Key points</h4>The volume-regulated anion channel (VRAC) is a swelling-activated chloride channel that is permeable to inorganic anions and a variety of small organic molecules. VRAC is formed via heteromerization of LRRC8 proteins, among which LRRC8A is essential, while LRRC8B/C/D/E serve as exchangeable complementary partners. We used an RNAi approach and radiotracer assays to explore which LRRC8 isoforms contribute to swelling-activated release of diverse organic osmolytes in rat astrocytes. Efflux of uncharged osmolytes (myo-inositol and taurine) was suppressed by deletion of LRRC8A or LRRC8D, but not by deletion of LRRC8C+LRRC8E. Conversely, release of charged osmolytes (d-aspartate) was strongly reduced by deletion of LRRC8A or LRRC8C+LRRC8E, but largely unaffected by downregulation of LRRC8D. Our findings point to the existence of multiple heteromeric VRACs in the same cell type: LRRC8A/D-containing heteromers appear to dominate release of uncharged osmolytes, while LRRC8A/C/E, with the additional contribution of LRRC8D, creates a conduit for movement of charged molecules.<h4>Abstract</h4>The volume-regulated anion channel (VRAC) is the ubiquitously expressed vertebrate Cl^- /anion channel that is composed of proteins belonging to the LRRC8 family and activated by cell swelling. In the brain, VRAC contributes to physiological and pathological release of a variety of small organic molecules, including the amino acid neurotransmitters glutamate, aspartate and taurine. In the present work, we explored the role of all five LRRC8 family members in the release of organic osmolytes from primary rat astrocytes. Expression of LRRC8 proteins was modified using an RNAi approach, and amino acid fluxes via VRAC were quantified by radiotracer assays in cells challenged with hypoosmotic medium (30% reduction in osmolarity). Consistent with our prior work, knockdown of LRRC8A potently and equally suppressed the release of radiolabelled d-[¹⁴ C]aspartate and [³ H]taurine. Among other LRRC8 subunits, downregulation of LRRC8D strongly inhibited release of the uncharged osmolytes [³ H]taurine and myo-[³ H]inositol, without major impact on the simultaneously measured efflux of the charged d-[¹⁴ C]aspartate. In contrast, the release of d-[¹⁴ C]aspartate was preferentially sensitive to deletion of LRRC8C+LRRC8E, but unaffected by downregulation of LRRC8D. Finally, siRNA knockdown of LRRC8C+LRRC8D strongly inhibited the release of all osmolytes. Overall, our findings suggest the existence of at least two distinct heteromeric VRACs in astroglial cells. The LRRC8A/D-containing permeability pathway appears to dominate the release of uncharged osmolytes, while an alternative channel (or channels) is composed of LRRC8A/C/D/E and responsible for the loss of charged molecules. << Less

  J. Physiol. (Lond.) 595:6939-6951(2017) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.