Light-Induced Depolarization of Neurons Using a Modified Shaker K+ Channel and a Molecular Photoswitch

Department of Molecular and Cell Biology , University of California, Berkeley, Berkeley, California, United States
Journal of Neurophysiology (Impact Factor: 2.89). 12/2006; 96(5):2792-6. DOI: 10.1152/jn.00318.2006
Source: PubMed


To trigger action potentials in neurons, most investigators use electrical or chemical stimulation. Here we describe an optical stimulation method based on semi-synthetic light-activated ion channels. These SPARK (synthetic photoisomerizable azobenzene-regulated K(+)) channels consist of a synthetic azobenzene-containing photoswitch and a genetically modified Shaker K(+) channel protein. SPARK channels with a wild-type selectivity filter elicit hyperpolarization and suppress action potential firing when activated by 390 nm light. A mutation in the pore converts the K(+)-selective Shaker channel into a nonselective cation channel. Activation of this modified channel with the same wavelength of light elicits depolarization of the membrane potential. Expression of these depolarizing SPARK channels in neurons allows light to rapidly and reversibly trigger action potential firing. Hence, hyper- and depolarizing SPARK channels provide a means for eliciting opposite effects on neurons in response to the same light stimulus.

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Available from: Matthew R Banghart, Apr 02, 2014
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    • "Exposure to ultraviolet light silenced spontaneous action potentials in transfected cells, which was restored upon visiblelight illumination. To photo stimulate action potentials, a Shaker pore mutation was used to convert the K + channel into a non-selective cation channel (Chambers et al. 2006). Later, MAL-AZO-QA was used to control the two-pore TREK1 channel in transfected hippocampal neurons (Sandoz et al. 2012). "
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    • "Sandoz and Levitz Optogenetics of potassium channels When overexpressed in cultured neurons, opening of D-SPARK can trigger light-dependent action potential firing (Chambers et al., 2006). While useful for photocontrol of neuronal activity, SPARK and D-SPARK are non-native and extensively mutated ion channels that do not permit one to study the role of specific potassium channels in neurons. "
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    • "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). "
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