Szobota Stephanie, McKenzie Catherine, Janovjak Harald
Department of Neurology, Yale University School of Medicine, New Haven, CT, USA.
Methods Mol Biol. 2013;998:417-35. doi: 10.1007/978-1-62703-351-0_32.
In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation of this approach are covalently attached photochromic tethered ligands (PTLs) that allow activating ligand-gated ion channels with outstanding spatial and temporal resolution. Here, we describe all steps towards the successful development and application of PTL-gated ion channels in cell lines and primary cells. The basis for these experiments forms a combination of molecular modeling, genetic engineering, cell culture, and electrophysiology. The light-gated glutamate receptor (LiGluR), which consists of the PTL-functionalized GluK2 receptor, serves as a model.
在充满活力的光遗传学领域，光学与基因靶向技术相结合，通过光刺激细胞膜中表达的光敏离子通道来调控细胞功能，如神经元动作电位。这种方法的一种广泛应用形式是共价连接的光致变色拴系配体（PTL），它能够以出色的空间和时间分辨率激活配体门控离子通道。在此，我们描述了在细胞系和原代细胞中成功开发和应用PTL门控离子通道的所有步骤。这些实验的基础是分子建模、基因工程、细胞培养和电生理学的结合。由PTL功能化的GluK2受体组成的光门控谷氨酸受体（LiGluR）用作模型。
