Department of Pharmacology, University of South Alabama, Mobile, AL, USA.
Center for Lung Biology, University of South Alabama, Mobile, AL, USA.
Methods Mol Biol. 2022;2483:265-279. doi: 10.1007/978-1-0716-2245-2_17.
In the last 20 years tremendous progress has been made in the development of single cell cAMP sensors. Sensors are based upon cAMP binding proteins that have been modified to transduce cAMP concentrations into electrical or fluorescent readouts that can be readily detected using patch clamp amplifiers, photomultiplier tubes, or cameras. Here, we describe two complementary approaches for the detection and measurement of cAMP signals near the plasma membrane of cells using cyclic nucleotide (CNG) channel-based probes. These probes take advantage of the ability of CNG channels to transduce small changes in cAMP concentration into ionic flux through channel pores that can be readily detected by measuring Ca and/or Mn influx or by measuring ionic currents.
在过去的 20 年中，单细胞 cAMP 传感器的开发取得了巨大进展。传感器基于已被修饰为将 cAMP 浓度转化为电或荧光读数的 cAMP 结合蛋白，这些读数可以使用膜片钳放大器、光电倍增管或相机轻松检测到。在这里，我们描述了两种使用基于环核苷酸 (CNG) 通道的探针检测和测量细胞靠近质膜处的 cAMP 信号的互补方法。这些探针利用 CNG 通道将 cAMP 浓度的微小变化转化为通过通道孔的离子通量的能力，通过测量 Ca 和/或 Mn 内流或测量离子电流可以很容易地检测到这种离子通量。
