Optogenetic photochemical control of designer K+ channels in mammalian neurons.

Currently available optogenetic tools, including microbial light-activated ion channels and transporters, are transforming systems neuroscience by enabling precise remote control of neuronal firing, but they tell us little about the role of indigenous ion channels in controlling neuronal function. Here, we employ a chemical-genetic strategy to engineer light sensitivity into several mammalian K(+) channels that have different gating and modulation properties. These channels provide the means for photoregulating diverse electrophysiological functions. Photosensitivity is conferred on a channel by a tethered ligand photoswitch that contains a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q), a K(+) channel pore blocker. Using mutagenesis, we identify the optimal extracellular cysteine attachment site where MAQ conjugation results in pore blockade when the azobenzene moiety is in the trans but not cis configuration. With this strategy, we have conferred photosensitivity on channels containing Kv1.3 subunits (which control axonal action potential repolarization), Kv3.1 subunits (which contribute to rapid-firing properties of brain neurons), Kv7.2 subunits (which underlie "M-current"), and SK2 subunits (which are Ca(2+)-activated K(+) channels that contribute to synaptic responses). These light-regulated channels may be overexpressed in genetically targeted neurons or substituted for native channels with gene knockin technology to enable precise optopharmacological manipulation of channel function.

The behavior of alpha-synuclein in neurons.

Despite considerable evidence linking alpha-synuclein with membranes in vitro, it has proven difficult to demonstrate membrane association of the protein in vivo. alpha-Synuclein localizes to the nerve terminal, but biochemical experiments have not revealed a tight association with membranes. To address the dynamics of the protein in live cells, we have used photobleaching and found that alpha-synuclein exhibits high mobility, although distinctly less than an entirely soluble protein. Further, neural activity controls the distribution of alpha-synuclein, causing its dispersion from the synapse. In addition to the presumed role of alpha-synuclein dynamics in synaptic function, changes in its physiological behavior may underlie the pathological changes associated with Parkinson's disease.

New photochemical tools for controlling neuronal activity.

Neurobiology has entered a new era in which optical methods are challenging electrophysiological techniques for their value in measuring and manipulating neuronal activity. This change is occurring largely because of the development of new photochemical tools, some synthesized by chemists and some provided by nature. This review is focused on the three types of photochemical tools for neuronal control that have emerged in recent years. Caged neurotransmitters, including caged glutamate, are synthetic molecules that enable highly localized activation of neurotransmitter receptors in response to light. Natural photosensitive proteins, including channelrhodopsin-2 and halorhodopsin, can be exogenously expressed in neurons and enable rapid photocontrol of action potential firing. Synthetic small molecule photoswitches can bestow light-sensitivity on native or exogenously expressed proteins, including K(+) channels and glutamate receptors, allowing photocontrol of action potential firing and synaptic events. At a rapid pace, these tools are being improved and new tools are being introduced, thanks to molecular biology and synthetic chemistry. The three families of photochemical tools have different capabilities and uses, but they all share in enabling precise and noninvasive exploration of neural function with light.

Photochemical control of endogenous ion channels and cellular excitability.

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.

Remote control of neuronal activity with a light-gated glutamate receptor.

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.

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling.

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.

Neural activity controls the synaptic accumulation of alpha-synuclein.

The presynaptic protein alpha-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. Alpha-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 alpha-synuclein in living neurons, we used fluorescence recovery after photobleaching. Despite its enrichment at the synapse, alpha-synuclein is highly mobile, with rapid exchange between adjacent synapses. In addition, we find that alpha-synuclein disperses from the nerve terminal in response to neural activity. Dispersion depends on exocytosis, but unlike other synaptic vesicle proteins, alpha-synuclein dissociates from the synaptic vesicle membrane after fusion. Furthermore, the dispersion of alpha-synuclein is graded with respect to stimulus intensity. Neural activity thus controls the normal function of alpha-synuclein at the nerve terminal and may influence its role in PD.

A combinatorial code for the interaction of alpha-synuclein with membranes.

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-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 alpha-synuclein. To understand how alpha-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 alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-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 alpha-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, alpha-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 alpha-synuclein with membranes.

Lipid rafts mediate the synaptic localization of alpha-synuclein.

Alpha-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, alpha-synuclein localizes almost exclusively to the nerve terminal. In brain extracts, however, alpha-synuclein behaves as a soluble, monomeric protein. Using a binding assay to characterize the association of alpha-synuclein with cell membranes, we find that alpha-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 alpha-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 alpha-synuclein with lipid rafts. Furthermore, we find that both the A30P mutation and raft disruption redistribute alpha-synuclein away from synapses, indicating an important role for raft association in the normal function of alpha-synuclein and its role in the pathogenesis of PD.