Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel
ABSTRACT Using light to silence electrical activity in targeted cells is a major goal of optogenetics. Available optogenetic proteins that directly move ions to achieve silencing are inefficient, pumping only a single ion per photon across the cell membrane rather than allowing many ions per photon to flow through a channel pore. Building on high-resolution crystal-structure analysis, pore vestibule modeling, and structure-guided protein engineering, we designed and characterized a class of channelrhodopsins (originally cation-conducting) converted into chloride-conducting anion channels. These tools enable fast optical inhibition of action potentials and can be engineered to display step-function kinetics for stable inhibition, outlasting light pulses and for orders-of-magnitude-greater light sensitivity of inhibited cells. The resulting family of proteins defines an approach to more physiological, efficient, and sensitive optogenetic inhibition.
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- "Second, engineering approaches aimed to increase light sensitivity and enhanced long-term photocurrent stability (similar to SFOs) cannot be applied to pumps efficiently, as they depend on pore size. Two excellent recent studies used the crystal structure of channelrhodopsin hybrid C1C2, determined by Kato et al. (2012), to create a class of light-activated inhibitory chloride channels (Berndt et al., 2014; Wietek et al., 2014). iC1C2 allows for blue-shifted inhibition with fast kinetics, while SwiChR provides step function Guru et al. | 5 inhibition. "
Article: Making sense of optogenetics[Show abstract] [Hide abstract]
ABSTRACT: This review, one of a series of articles, tries to make sense of optogenetics, a recently developed technology that can be used to control the activity of genetically defined neurons with light. Cells are first genetically engineered to express a light-sensitive opsin, which is typically an ion channel, pump, or G protein-coupled receptor. When engineered cells are then illuminated with light of the correct frequency, opsin-bound retinal undergoes a conformational change that leads to channel opening or pump activation, cell depolarization or hyperpolarization, and neural activation or silencing. Since the advent of optogenetics many different opsin variants have been discovered or engineered, and it is now possible to stimulate or inhibit neuronal activity or intracellular signaling pathways on fast or slow timescales with a variety of different wavelengths of light. Optogenetics has been successfully employed to enhance our understanding of the neural circuit dysfunction underlying mood disorders, addiction, and Parkinson's disease, and has enabled us to achieve a better understanding of the neural circuits mediating normal behavior. It has revolutionized the field of neuroscience, and has enabled a new generation of experiments that probe the causal roles of specific neural circuit components. © The Author 2015. Published by Oxford University Press on behalf of CINP.The International Journal of Neuropsychopharmacology 07/2015; DOI:10.1093/ijnp/pyv079 · 5.26 Impact Factor
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- "Perhaps most importantly, many of these links remain purely correlative, and it will be critical to test whether and how changes in synaptic remodeling directly affect the function of cortical microcircuits , the integration of information across neuroanatomically distributed networks, and the emergence of behavioral effects and psychiatric symptoms. To this end, the recent development of optogenetic tools for manipulating activity in specific neural circuits will be critical for establishing causal mechanisms (Yizhar et al., 2011; Tye and Deisseroth, 2012; Berndt et al., 2014). Likewise , recently developed imaging modalities provide a means for testing how structural changes within a given microcircuit affect functional circuit dynamicsdanother critical, unanswered question . "
ABSTRACT: Stress-especially chronic, uncontrollable stress-is an important risk factor for many neuropsychiatric disorders. The underlying mechanisms are complex and multifactorial, but they involve correlated changes in structural and functional measures of neuronal connectivity within cortical microcircuits and across neuroanatomically distributed brain networks. Here, we review evidence from animal models and human neuroimaging studies implicating stress-associated changes in functional connectivity in the pathogenesis of PTSD, depression, and other neuropsychiatric conditions. Changes in fMRI measures of corticocortical connectivity across distributed networks may be caused by specific structural alterations that have been observed in the prefrontal cortex, hippocampus, and other vulnerable brain regions. These effects are mediated in part by glucocorticoids, which are released from the adrenal gland in response to a stressor and also oscillate in synchrony with diurnal rhythms. Recent work indicates that circadian glucocorticoid oscillations act to balance synapse formation and pruning after learning and during development, and chronic stress disrupts this balance. We conclude by considering how disrupted glucocorticoid oscillations may contribute to the pathophysiology of depression and PTSD in vulnerable individuals, and how circadian rhythm disturbances may affect non-psychiatric populations, including frequent travelers, shift workers, and patients undergoing treatment for autoimmune disorders.01/2015; 1:174-183. DOI:10.1016/j.ynstr.2014.10.008
- Science 04/2014; 344(6182):369-70. DOI:10.1126/science.1253616 · 31.48 Impact Factor