Coupling of simultaneous fluorescence and electrophysiology: historical survey, contributions, and prospects

The coupling of simultaneous fluorescence and electrophysiology measurements paved the way for the development of potentiometric and intracellular free ion-sensitive probes, although the basic and initial aim of these combined studies remains to record fast conformational changes during ion channel activation. Recent high-resolution studies of some channels put back on the agenda challenging methods that combine spectroscopy and electrophysiology. Fluorescence is certainly the most versatile and sensitive spectroscopy in this kind of experiment, and we have recently witnessed significant breakthroughs, at the level of whole cells with intact or mutated channels or with planar lipid bilayers doped with channels or their peptide models. After our initial study of membrane dynamics associated with excitability, following transient pyrene excimer signals during action potentials or voltage-clamp of nerve fibers, we tested the feasibility of FRAP (fluorescence recovery after photobleaching) experiments on planar lipid bilayers. This technique was later applied to lateral diffusion measurements of channel forming peptides (alamethicin labeled with fluorescein and the voltage-sensitive segment S4 of the Shaker potassium channel labeled with NBD) under applied voltage. We provide independent evidence for voltage-dependent partitioning and transmembrane insertion and propose renewed experimental avenues to reveal movements and conformational changes associated with ion channel gating and opening.