Neurons possess ion channels that are directly sensitive to voltage, ligands, temperature, or mechanical forces, but not to light. To allow optical regulation of neural activity, Kramer and colleagues have used a combination of organic chemistry and molecular biology to engineer ion channels that are directly opened and closed with different wavelengths of light (Banghart et al., 2004). These "hybrid" channels have two parts: a synthetic photoswitch molecule and a specially modified Shaker K+ channel protein. The photoswitch molecule (MAL-AZO-QA) contains a cysteine-reactive maleimide group (MAL), for covalent attachment to the channel protein, a photoisomerizable azobenzene group (AZO), which changes length in response to light, and a quaternary ammonium ion (QA), with blocks the pore of K+ channels (Fig. 8A). Various engineered mutations in the Shaker K+ channel minimize inactivation and shift the voltage dependence of activation, rendering the channel constitutively open in the absence of the photoswitch. The channel also has a cysteine engineered into a key extracellular location, at which the photo-switch can covalently attach to the channel, thus tethering the QA group near the pore of the channel. In the dark or in visible light (>450 nm), the AZO group is in its extended trans configuration, and the QA can reach the pore and block conduction of K+ ions. Exposure to near-UV light (380 nm) photoisomerizes the AZO to its bent cis-configuration, thereby shortening the molecule so that the QA cannot reach the pore, which then permits conduction of K+ ions. Thus, different wavelengths of light open or close the channel by advancing or retracting the blocking group from the pore.
This synthetic photoisomerizable azobenzene-regulated K+ channel (called the "SPARK" channel) can be exogenously expressed in various cell types with standard gene transfection methods. Light opens and closes SPARK channels within seconds, and it can be used repeatedly with little or no decrement in the response (Fig. 8B). In hippocampal neurons, expression of the modified Shaker channel, followed by treatment with MAL-AZO-QA, generates SPARK channels that can be used for photic activation of the K+ conductance and thus can reversibly suppress action potential firing (Fig. 8C). Remarkably, MAL-AZO-QA has no detectable effect on neurons that fail to express the Shaker channel. This is because successful coupling of MAL-AZO-QA with the channel requires that the distance between the cysteine attachment site and the QA binding site in the pore to closely match the length of the photoswitch molecule. The specificity of the photoswitch for the exogenously expressed Shaker channel implies that SPARK channels can be targeted to specific types of neurons simply by targeting Shaker channel expression with cell-type-specific promoters.
Because the photoswitch is covalently attached to an ion channel, which is integral to the plasma membrane of the targeted neuron, this approach avoids the use of diffusible chemical activators that can limit the spatial and temporal accuracy of photic control of neuronal activity. The structural basis of channel function is perhaps better understood for Shaker than for any other voltage-gated channel (Jan and Jan, 1997), making it easy to customize SPARK for particular uses by altering the channel protein. Thus, mutations can be made in the pore-lining domain to convert the K+-selective Shaker channel into a nonselective cation channel (Heginbotham et al., 1994), changing the channel from one that hyperpolarizes cells when it is opened with light (H-SPARK) into a channel that depolarizes (D-SPARK). Introduction of a specific dendritic targeting sequence (Rivera et al., 2003) directs SPARK channels to the dendritic tree, allowing optical regulation of dendritic spiking. The photoswitch molecule can also be modified, for example, to change the stability of the cis- configuration, resulting in persistent SPARK channel activation triggered by a brief flash of light.
The creation of SPARK channels provides a precise and non-invasive way to control neural activity, with experimental and, perhaps eventually, medical applications. Acute activation of SPARK channels, either in single neurons or many neurons of a given type, can be used to assess the role of neurons in complex neural circuits. Sustained activation of SPARK channels, for example, in the dendritic tree, can be used to investigate homeostasis of neuronal excitability. Farther down the road is the possibility of introducing SPARK channels in vivo by transgenesis or viral transduction, to allow light to control activity in intact neural structures. The most intriguing possibility is the retina, the one part of the nervous system normally accessible to light. By expressing D-SPARK and H-SPARK channels in retinal ganglion cells, it may be possible to generate virtual "On" and "Off" cells that are directly activated or inhibited by light. This could enable light to directly control spiking in these cells, even if the natural photoreceptors (the rods and cones) have degenerated because of experimental manipulations or disease.
In conclusion, the data discussed here demonstrate that light is not merely a medium for passively visualizing structures and events but rather a tool to be used actively for manipulating those structures and events. The approaches described above represent enabling technology that has wide application in neurobiology. We are convinced that the introduction of this powerful new technology will invariably stimulate new, as well as new types of, experiments.
Received Aug 19, 2005; revised September 1, 2005; accepted September 1, 2005.
This work was supported by grants from the National Institutes of Health (MH65488 and NS40338 to S.M.T., GM56481 and GM64706 to J.P.Y.K., EY 12649 to R.H.K., MH072698 to Graeme Davis, and NS045193 to S.S.-H.W.) and the National Science Foundation (IOB 0347719 to S.S.-H.W.), by a Keck Foundation Young Investigator Award (S.S.-H.W.), and by a Wellcome Trust Senior Fellowship (R.A.S.).
Correspondence should be addressed to Scott Thompson, Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201. E-mail: [email protected]
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