Optogenetic photochemical control of designer K+channels in mammalian neurons
| Autor: | Matthew R. Banghart, Alexis Fedorchak, Duane Allen, Doris L. Fortin, Dirk Trauner, John P. Adelman, Rachel Montpetit, Richard H. Kramer, Timothy W. Dunn |
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| Rok vydání: | 2011 |
| Předmět: |
Physiology
Molecular Sequence Data Gating Optogenetics Protein Engineering Maleimides Potassium Channels Calcium-Activated Humans KCNQ2 Potassium Channel Repolarization Amino Acid Sequence Ion channel Neurons Kv1.3 Potassium Channel Photoswitch Chemistry General Neuroscience Light-gated ion channel Photochemical Processes Potassium channel Quaternary Ammonium Compounds Electrophysiology HEK293 Cells nervous system Innovative Methodology Biophysics Azo Compounds Ion Channel Gating |
| Zdroj: | Journal of Neurophysiology. 106:488-496 |
| ISSN: | 1522-1598 0022-3077 |
| Popis: | 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 Ca2+-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. |
| Databáze: | OpenAIRE |
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