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.
"Fortin et al. (2011) proposed to use K V 7.2 to control the resting membrane potential, because it has a low activation threshold and Kv3.1 to control accommodation because these channels have a high-threshold of activation and activate and deactivate rapidly (Gan and Kaczmarek, 1998). Most notably, photoswitchable SK2 channels were used to control the size of EPSPs in CA1 hippocampal neurons where they are natively involved in dendritic repolarization following glutamate receptor-mediated depolarization (Fortin et al., 2011). "
[Show abstract][Hide abstract] ABSTRACT: Optogenetic tools were originally designed to target specific neurons for remote control of their activity by light and have largely been built around opsin-based channels and pumps. These naturally photosensitive opsins are microbial in origin and are unable to mimic the properties of native neuronal receptors and channels. Over the last 8 years, photoswitchable tethered ligands (PTLs) have enabled fast and reversible control of mammalian ion channels, allowing optical control of neuronal activity. One such PTL, maleimide-azobenzene-quaternary ammonium (MAQ), contains a maleimide (M) to tether the molecule to a genetically engineered cysteine, a photoisomerizable azobenzene (A) linker and a pore-blocking quaternary ammonium group (Q). MAQ was originally used to photocontrol SPARK, an engineered light-gated potassium channel derived from Shaker. Potassium channel photoblock by MAQ has recently been extended to a diverse set of mammalian potassium channels including channels in the voltage-gated and K2P families. Photoswitchable potassium channels, which maintain native properties, pave the way for the optical control of specific aspects of neuronal function and for high precision probing of a specific channel's physiological functions. To extend optical control to natively expressed channels, without overexpression, one possibility is to develop a knock-in mouse in which the wild-type channel gene is replaced by its light-gated version. Alternatively, the recently developed photoswitchable conditional subunit technique provides photocontrol of the channel of interest by molecular replacement of wild-type complexes. Finally, photochromic ligands also allow photocontrol of potassium channels without genetic manipulation using soluble compounds. In this review we discuss different techniques for optical control of native potassium channels and their associated advantages and disadvantages.
"The photoactivation process can be made more efficient by linking the ligand to the protein through a covalent bond and obtaining a " photoswitched tethered ligand " (PTL, Fig. 2a), a technique used successfully to control nicotinic receptors (Bartels et al., 1971), ionotropic glutamate receptors (Volgraf et al., 2006), potassium channels (Banghart et al., 2004; Chambers et al., 2006; Fortin et al., 2011), and recently a chimeric potassium-selective glutamate receptor called HyLighter (Janovjak et al., 2010). One major drawback of photochemical approaches is the necessity of either delivering the ligand or conjugating the PTL to the target protein, which limits their use to easily accessible preparations like cultured neurons, brain slices, or small organisms such as fruit flies (Lima and Miesenböck, 2005) or zebrafish larvae (Janovjak et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: Fundamental questions that neuroscientists have previously approached with classical biochemical and electrophysiological techniques can now be addressed using optogenetics. The term optogenetics reflects the key program of this emerging field, namely, combining optical and genetic techniques. With the already impressively successful application of light-driven actuator proteins such as microbial opsins to interact with intact neural circuits, optogenetics rose to a key technology over the past few years. While spearheaded by tools to control membrane voltage, the more general concept of optogenetics includes the use of a variety of genetically encoded probes for physiological parameters ranging from membrane voltage and calcium concentration to metabolism. Here, we provide a comprehensive overview of the state of the art in this rapidly growing discipline and attempt to sketch some of its future prospects and challenges.
Progress in brain research 01/2012; 196:1-28. DOI:10.1016/B978-0-444-59426-6.00001-X · 2.83 Impact Factor
"While the popularity of these reengineered channels suffers from the fact that they involve additional, potentially toxic steps to implement (conjugation of tethered ligands), they may still be useful for some applications (Gorostiza and Isacoff, 2007; Chambers and Kramer, 2008). In fact, Kv3.1-, Kv7.2-, and SK2-containing potassium currents, which do not have specific pharmacological blockers, have been tethered to a quaternary ammonia potassium channel blocker with a photoisomerizable azobenzene (Fortin et al., 2011). These modified K+ channels can be specifically and reversibly manipulated, a feat not possible with pharmacology alone. "
[Show abstract][Hide abstract] ABSTRACT: Optical manipulation of neuronal activity has rapidly developed into the most powerful and widely used approach to study mechanisms related to neuronal connectivity over a range of scales. Since the early use of single site uncaging to map network connectivity, rapid technological development of light modulation techniques has added important new options, such as fast scanning photostimulation, massively parallel control of light stimuli, holographic uncaging, and two-photon stimulation techniques. Exciting new developments in optogenetics complement neurotransmitter uncaging techniques by providing cell-type specificity and in vivo usability, providing optical access to the neural substrates of behavior. Here we review the rapid evolution of methods for the optical manipulation of neuronal activity, emphasizing crucial recent developments.
Frontiers in Systems Neuroscience 12/2011; 5:95. DOI:10.3389/fnsys.2011.00095
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