Chernyshkova Olga V, Belyakov Mikhail V, Meshalkina Darya A, Firsov Mikhail L
Sechenov Institute of Evolutional Physiology and Biochemistry, RAS, St. Petersburg, Russia.
Research Institute of Hygiene, Occupational Pathology and Human Ecology of the Federal Medical-Biological Agency, St. Petersburg, Russia.
Bio Protoc. 2025 Sep 5;15(17):e5431. doi: 10.21769/BioProtoc.5431.
The phototransduction cascade allows photoreceptors to detect light across a wide range of intensities without saturation, with cGMP serving as the second messenger and calcium feedback as the key regulatory mechanism. While experimental evidence suggests that cAMP may also play a role in modulating this cascade, such regulation would necessitate rapid changes in cAMP levels on a timescale of seconds. However, data on the dynamics of intracellular cAMP changes in photoreceptors remain scarce, primarily due to the limitations of conventional fluorescence-based methods in this specialized sensory system. To address this gap, we developed a methodology combining rapid cryofixation of retinal samples following light stimulation with the isolation of outer segment preparations. The rapid cryofixation setup comprises six computer-controlled sections, each with a high-speed stepper motor-driven lever that rapidly moves the specimen in a 180° arc within ~80 ms to press it against a liquid nitrogen-cooled copper cylinder for fixation. Using highly sensitive metabolomics techniques, we measured cAMP levels in these samples. This approach enables the investigation of rapid cAMP dynamics and its potential regulatory role in phototransduction, providing a foundation for understanding the interplay between cAMP and PKA signaling in photoreceptor function. Key features • The protocol provides ms time resolution in retina outer segment sampling in response to light stimulus with cryofixation, conserving proteome and metabolome response features. • The protocol allows direct cAMP quantification with an average level of 11.4 ± 0.5 pmol/mg of protein in the dark.
光转导级联反应使光感受器能够在很宽的强度范围内检测光而不饱和，其中环磷酸鸟苷（cGMP）作为第二信使，钙反馈作为关键调节机制。虽然实验证据表明环磷酸腺苷（cAMP）也可能在调节这一级联反应中发挥作用，但这种调节需要cAMP水平在数秒的时间尺度上快速变化。然而，关于光感受器细胞内环磷酸腺苷变化动力学的数据仍然很少，主要是由于在这个特殊的感觉系统中传统荧光方法存在局限性。为了填补这一空白，我们开发了一种方法，将光刺激后视网膜样本的快速冷冻固定与分离外段制备物相结合。快速冷冻固定装置包括六个由计算机控制的部分，每个部分都有一个由高速步进电机驱动的杠杆，该杠杆在约80毫秒内将标本在180°弧内快速移动，将其压在液氮冷却的铜柱上进行固定。我们使用高度灵敏的代谢组学技术测量了这些样本中的cAMP水平。这种方法能够研究快速的cAMP动力学及其在光转导中的潜在调节作用，为理解cAMP和蛋白激酶A（PKA）信号在光感受器功能中的相互作用奠定了基础。关键特性•该方案在视网膜外段采样中对光刺激响应进行冷冻固定时提供毫秒级时间分辨率，保留蛋白质组和代谢组响应特征。•该方案允许直接定量cAMP，在黑暗中平均水平为11.4±0.5皮摩尔/毫克蛋白质。
