Optogenetic photochemical control of designer K+ channels in mammalian neurons
Optogenetic photochemical control of designer K+ channels in mammalian neurons
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DOI：
10.1152/jn.00251.2011
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发表时间：
2011-07-01
影响因子：
2.5
通讯作者：
Kramer, Richard H.
中科院分区：
文献类型：
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作者：
Fortin, Doris L.;Dunn, Timothy W.;Kramer, Richard H.
Fortin DL, Dunn TW, Fedorchak A, Allen D, Montpetit R, Banghart MR, Trauner D, Adelman JP, Kramer RH。哺乳动物神经元中设计K+通道的光遗传光化学控制。[J]中国生物医学工程学报，2016,31(4):559 - 567。首次发表于2011年4月27日；doi: 10.1152 / jn.00251.2011。-目前可用的光遗传学工具，包括微生物光激活离子通道和转运体，正在通过精确远程控制神经元放电来改变系统神经科学，但它们很少告诉我们原生离子通道在控制神经元功能中的作用。在这里，我们采用化学遗传策略来设计具有不同门控和调制特性的几种哺乳动物K+通道的光敏性。这些通道提供了光调节各种电生理功能的手段。光敏性通过拴链配体光开关赋予通道，该配体光开关包含半胱氨酸反应性马来酰亚胺(M)、光异构偶氮苯(a)和K+通道孔阻滞剂季铵(Q)。利用诱变技术，我们确定了最佳的细胞外半胱氨酸附着位点，当偶氮苯部分处于反式而不是顺式构型时，MAQ偶联会导致孔阻塞。通过这种策略，我们将光敏性赋予了含有Kv1.3亚基（控制轴突动作电位复极化）、Kv3.1亚基（有助于大脑神经元的快速放电特性）、Kv7.2亚基（构成“m电流”的基础）和SK2亚基（Ca2+激活的K+通道，有助于突触反应）的通道。这些光调节通道可能在基因靶向神经元中过度表达，或者用基因敲入技术取代天然通道，从而实现对通道功能的精确光药理学操作。
Fortin DL, Dunn TW, Fedorchak A, Allen D, Montpetit R, Banghart MR, Trauner D, Adelman JP, Kramer RH. Optogenetic photochemical control of designer K+ channels in mammalian neurons. J Neurophysiol 106: 488-496, 2011. First published April 27, 2011; doi:10.1152/jn.00251.2011.-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.