The quantal release of glutamate depends on its transport into synaptic vesicles. Recent work has shown that a protein previously implicated in the uptake of inorganic phosphate across the plasma membrane catalyzes glutamate uptake by synaptic vesicles. However, only a subset of glutamate neurons expresses this vesicular glutamate transporter (VGLUT1). We now report that excitatory neurons lacking VGLUT1 express a closely related protein that has also been implicated in phosphate transport. Like VGLUT1, this protein localizes to synaptic vesicles and functions as a vesicular glutamate transporter (VGLUT2). The complementary expression of VGLUT1 and 2 defines two distinct classes of excitatory synapse.
Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.
␣-Synuclein contributes to the pathogenesis of Parkinson's disease (PD), but its precise role in the disorder and its normal function remain poorly understood. Consistent with a presumed role in neurotransmitter release and its prominent deposition in the dystrophic neurites of PD, ␣-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, ␣-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of ␣-synuclein with cell membranes, we find that ␣-synuclein binds saturably and with high affinity to characteristic intracellular structures that double label for components of lipid rafts. Biochemical analysis demonstrates the interaction of ␣-synuclein with detergent-resistant membranes and reveals a shift in electrophoretic mobility of the raft-associated protein. In addition, the A30P mutation associated with PD disrupts the interaction of ␣-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute ␣-synuclein away from synapses, indicating an important role for raft association in the normal function of ␣-synuclein and its role in the pathogenesis of PD.
The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.
The presynaptic protein ␣-synuclein has a central role in Parkinson's disease (PD). However, the mechanism by which the protein contributes to neurodegeneration and its normal function remain unknown. ␣-Synuclein localizes to the nerve terminal and interacts with artificial membranes in vitro but binds weakly to native brain membranes. To characterize the membrane association of ␣-synuclein in living neurons, we used fluorescence recovery after photobleaching. Despite its enrichment at the synapse, ␣-synuclein is highly mobile, with rapid exchange between adjacent synapses. In addition, we find that ␣-synuclein disperses from the nerve terminal in response to neural activity. Dispersion depends on exocytosis, but unlike other synaptic vesicle proteins, ␣-synuclein dissociates from the synaptic vesicle membrane after fusion. Furthermore, the dispersion of ␣-synuclein is graded with respect to stimulus intensity. Neural activity thus controls the normal function of ␣-synuclein at the nerve terminal and may influence its role in PD.
Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small molecule, or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.
Light work: Studies into the mechanism of AAQ, a photoswitchable blocker of voltage‐gated K+ channels, led to the discovery and development of photochromic ligands that act at the internal tetraethylammonium binding site (see picture). These molecules can be applied from the extracellular side to impart long‐lasting photosensitivity on K+ channels in living cells and thereby afford photocontrol of action potential firing in neurons.
Considerable genetic and pathological evidence has implicated the small, soluble protein ␣-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of ␣-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of ␣-synuclein. To understand how ␣-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant ␣-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of ␣-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that ␣-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of ␣-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with ␣-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, ␣-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of ␣-synuclein with membranes.
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