Abstract
Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.
Highlights
With the discovery of channelrhodopsin-1 (Nagel et al, 2002) and the demonstration of lightinduced membrane depolarization via ChR2 (Nagel et al, 2003), optical manipulation of cell physiology with transgenic photoreceptors became the method of choice for manipulating genetically defined cells (Boyden et al, 2005; Li et al, 2005; Nagel et al, 2005; Bi et al, 2006); Optogenetic Activation and Inhibition Tools (Ishizuka et al, 2006)
Highly efficient Cl− conducting anion channelrhodopsins (ACRs) have been introduced (Govorunova et al, 2015), but whether they hyperpolarize or depolarize cells depends on the intracellular Cl− concentration (Mahn et al, 2016; Wiegert and Oertner, 2016)
Illumination led to body contraction with the OLF fusion construct, and to body extension with the SthK fusion construct
Summary
With the discovery of channelrhodopsin-1 (Nagel et al, 2002) and the demonstration of lightinduced membrane depolarization via ChR2 (Nagel et al, 2003), optical manipulation of cell physiology with transgenic photoreceptors became the method of choice for manipulating genetically defined cells (Boyden et al, 2005; Li et al, 2005; Nagel et al, 2005; Bi et al, 2006); Optogenetic Activation and Inhibition Tools (Ishizuka et al, 2006). The opsin-based toolbox has expanded and includes the earlier discovered and characterized pump rhodopsins (Zhang et al, 2007; Chow et al, 2010), engineered channel rhodopsins (Kleinlogel et al, 2011; Lin et al, 2013; Dawydow et al, 2014; Scholz et al, 2017), and more recently, light-gated anion channels and nucleotidyl cyclases (Klapoetke et al, 2014; Gao et al, 2015; Govorunova et al, 2015; Scheib et al, 2015). The first optogenetic application employing a light-activated enzyme was light-induced increase of cytosolic cAMP with the photoactivated adenylyl cyclases PACα and PACβ (SchröderLang et al, 2007). These flavoproteins with a BLUF domain (blue light using FAD) were discovered in the unicellular flagellate Euglena gracilis (Iseki et al, 2002). In the genome of the soil bacterium Beggiatoa, a smaller photoactivated adenylyl cyclase (bPAC) was found and characterized (Ryu et al, 2010; Stierl et al, 2011)
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