From biophysics to neuroscience tools The channelrhodopsins and their distinctive light-activated ion channels have emerged as major tools in modern biological research. Deisseroth and Hegemann review the structural and functional properties of these protein photoreceptors. Mutagenesis and modeling studies, coupled with the reintroduction of modified channels into living systems, offer a profound understanding of how these channels work. The insights into the underlying basic science provide foundations for developing further applications in biology and medicine. Science, this issue p. eaan5544 BACKGROUND Channelrhodopsins (ChRs) are naturally occurring light-gated ion channels that are important for allowing motile algal cells to find suitable light levels. In neuroscience, ChRs have become broadly significant for helping to enable the control of specific circuit elements with light (i.e., optogenetics). Research into how sensation, cognition, and behavior arise from neuronal activity dynamics has been enabled by the expression of ChRs, and other members of the microbial opsin family, in specific cells or in specific connections within nervous systems of behaving animals. Both the unique light-gated channels themselves and opportunities for their biological application have been under intense investigation. The resulting studies of atomic-scale structure-function relationships have led not only to sophisticated understanding of the underlying chemical processes governing these unique seven-transmembrane channels from the plant kingdom, but also (via optogenetics) to the discovery of fundamental neural circuit principles underlying adaptive and maladaptive behavior in animals. ADVANCES The atomic-scale understanding of light-gated ion channel function has spanned the key processes of activation/deactivation gating, light adaptation, color tuning, and ion selectivity. A ChR crystal structure–derived, molecular dynamics–calculated pore snapshot (top left panel of the figure) summarizes the wide scope of biophysical and biochemical discoveries. Molecular modeling and redesign have created multiple modes of coupling between delivered photons and spikes in an approach that has illuminated basic principles of protein function and also created new tools for optogenetics. In the top right panel of the figure, the top trace shows a photon-spike transduction mode arising from the ChETA mutation, which results in high-speed, high-fidelity single blue flash–single spike coupling. The second trace shows red photon-spike transduction arising from a redshifted ChR found in nature and then engineered for stronger, more redshifted performance (C1V1). The third trace shows bistable excitation photon-spike logic, in which step-function opsin (SFO) mutations were introduced to create stalled photocycles, allowing stable excitation without continuous light delivery. The bottom trace shows bistable inhibition photon-spike logic; ChRs that are normally cation-conducting, and are therefore excitatory in neural systems, were converted to anion-conducting (inhibitory) ChRs by replacing negatively charged pore residues, followed by SFO mutations for bistability. The C1V1 and SFO designs together allowed us to determine that the medial prefrontal neocortex modulates interactions between two distant subcortical structures to control reward-mediating physiology and behavior (clarityresourcecenter.org/ofMRI.html; www.optogenetics.org). OUTLOOK The ChR light-gated pore will continue to be studied for its own elegant properties, which are paradigmatic among ion channels because light-gated systems allow structure-function analysis on the femtosecond time scale. Meanwhile, psychiatry has already yielded some of its deepest mysteries to ChR pore structural insights, including in explorations of clinically relevant behavioral states such as anhedonia. Many more opportunities for ChRs in basic neuroscience remain untapped, with the potential for precision redesign to achieve new applications and new roles integrated with other advanced technologies. A light-gated ion pore. (Top) Left: Inner workings of channelrhodopsin. Right: New photon-spike transduction modes arising from structure-guided redesign. (Bottom) Discovering the causal underpinnings of depression-related symptomatology. Brain region–specific activity dynamics of the mammalian dopamine neuron–driven reward state (left) are suppressed by the prefrontal cortex (right) as shown, using the second and third photon-spike transduction modes. Channelrhodopsins are light-gated ion channels that, via regulation of flagellar function, enable single-celled motile algae to seek ambient light conditions suitable for photosynthesis and survival. These plant behavioral responses were initially investigated more than 150 years ago. Recently, major principles of function for light-gated ion channels have been elucidated by creating channelrhodopsins with kinetics that are accelerated or slowed over orders of magnitude, by discovering and designing channelrhodopsins with altered spectral properties, by solving the high-resolution channelrhodopsin crystal structure, and by structural model–guided redesign of channelrhodopsins for altered ion selectivity. Each of these discoveries not only revealed basic principles governing the operation of light-gated ion channels, but also enabled the creation of new proteins for illuminating, via optogenetics, the fundamentals of brain function.

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通道视紫红质的形式和功能

从生物物理学到神经科学工具通道视紫红质及其独特的光激活离子通道已成为现代生物学研究的主要工具。Deisseroth 和 Hegemann 回顾了这些蛋白质光感受器的结构和功能特性。诱变和建模研究，再加上将经过修饰的通道重新引入生命系统，可以让人们对这些通道的工作原理有深刻的了解。对基础科学的深入了解为开发生物学和医学中的进一步应用奠定了基础。科学，这个问题 p。eaan5544 背景 通道视紫红质 (ChRs) 是天然存在的光门控离子通道，对于允许运动的藻类细胞找到合适的光照水平很重要。在神经科学中，ChRs 已经变得广泛重要，可以帮助用光控制特定的电路元件（即光遗传学）。通过 ChR 和微生物视蛋白家族的其他成员在行为动物的特定细胞或神经系统内的特定连接中的表达，研究了感觉、认知和行为如何从神经元活动动力学中产生。独特的光门控通道本身及其生物学应用的机会都在深入研究中。对原子级结构-功能关系的研究成果不仅使人们对控制植物界这些独特的七次跨膜通道的潜在化学过程有了深入的了解，而且（通过光遗传学）还发现了动物适应性和适应不良行为背后的基本神经回路原理。进展 对光门控离子通道功能的原子级理解已经跨越了激活/去激活门控、光适应、颜色调谐和离子选择性等关键过程。ChR 晶体结构衍生、分子动力学计算的孔隙快照（图的左上图）总结了广泛的生物物理和生物化学发现。分子建模和重新设计在传递的光子和尖峰之间创建了多种耦合模式，这种方法阐明了蛋白质功能的基本原理，还为光遗传学创建了新工具。在图中的右上面板中，顶部轨迹显示由 ChETA 突变引起的光子尖峰转导模式，这导致高速、高保真度的单蓝色闪光-单尖峰耦合。第二条迹线显示了自然界中发现的红移 ChR 产生的红色光子尖峰转导，然后经过工程设计以获得更强、更红移的性能 (C1V1)。第三条迹线显示了双稳态激发光子尖峰逻辑，其中引入了阶跃函数视蛋白 (SFO) 突变以产生停滞的光循环，从而无需连续光传输即可实现稳定激发。底部迹线显示双稳态抑制光子尖峰逻辑；通常是阳离子传导的 ChR，因此在神经系统中具有兴奋性，通过替换带负电荷的孔残基，然后进行双稳态 SFO 突变，转化为阴离子传导（抑制性）ChR。C1V1 和 SFO 设计共同使我们能够确定内侧前额叶新皮质调节两个远距离皮质下结构之间的相互作用，以控制奖赏调节生理学和行为（clarityresourcecenter.org/ofMRI.html；www.optogenetics.org）。展望 ChR 光门控孔将继续研究其自身的优雅特性，这些特性在离子通道中是典型的，因为光门控系统允许在飞秒时间尺度上进行结构-功能分析。与此同时，精神病学已经揭示了 ChR 孔结构洞察力的一些最深奥的谜团，包括对临床相关行为状态（如快感缺乏）的探索。ChRs 在基础神经科学中还有更多机会尚未开发，具有精确重新设计的潜力，以实现与其他先进技术集成的新应用和新角色。光门控离子孔。（上）左：通道视紫红质的内部运作。右图：由结构引导的重新设计产生的新光子尖峰转换模式。（下）发现抑郁症相关症状的因果基础。哺乳动物多巴胺神经元驱动的奖赏状态（左）的大脑区域特定活动动力学被前额皮质（右）抑制，如图所示，使用第二和第三光子尖峰转导模式。Channelrhodopsins 是光门控离子通道，通过调节鞭毛功能，使单细胞运动藻类能够寻找适合光合作用和生存的环境光条件。这些植物行为反应最初是在 150 多年前进行的。最近，光门控离子通道的主要功能原理已经通过创建具有在数量级上加速或减慢的动力学的通道视紫红质，通过发现和设计具有改变的光谱特性的通道视紫红质，通过解决高分辨率通道视紫红质晶体结构，并通过结构模型指导重新设计通道视紫红质以改变离子选择性。这些发现中的每一个不仅揭示了控制光门控离子通道运行的基本原理，而且还促成了新蛋白质的产生，以通过光遗传学阐明大脑功能的基本原理。光门控离子通道的主要功能原理已经通过创建具有加速或减慢几个数量级的动力学的通道视紫红质，通过发现和设计具有改变的光谱特性的通道视紫红质，通过解决高分辨率通道视紫红质晶体结构，以及通过结构模型指导的通道视紫红质重新设计以改变离子选择性。这些发现中的每一个不仅揭示了控制光门控离子通道运行的基本原理，而且还促成了新蛋白质的产生，以通过光遗传学阐明大脑功能的基本原理。光门控离子通道的主要功能原理已经通过创建具有加速或减慢几个数量级的动力学的通道视紫红质，通过发现和设计具有改变的光谱特性的通道视紫红质，通过解决高分辨率通道视紫红质晶体结构，以及通过结构模型指导的通道视紫红质重新设计以改变离子选择性。这些发现中的每一个不仅揭示了控制光门控离子通道运行的基本原理，而且还促成了新蛋白质的产生，以通过光遗传学阐明大脑功能的基本原理。通过解决高分辨率通道视紫红质晶体结构，并通过结构模型引导的通道视紫红质重新设计以改变离子选择性。这些发现中的每一个不仅揭示了控制光门控离子通道运行的基本原理，而且还促成了新蛋白质的产生，以通过光遗传学阐明大脑功能的基本原理。通过解决高分辨率通道视紫红质晶体结构，并通过结构模型引导的通道视紫红质重新设计以改变离子选择性。这些发现中的每一个不仅揭示了控制光门控离子通道运行的基本原理，而且还促成了新蛋白质的产生，以通过光遗传学阐明大脑功能的基本原理。