A non-covalently bound fluorescent probe with a larger fluorescence change for imaging Kv7 channels

The dysfunction of Kv7 channels (KCNQ channels) can lead to epilepsy, deafness, neurodevelopmental disorders and mental retardation. Monitoring the expression and function of Kv7 channels is essential for understanding their roles in physiological and pathological process. Some small-molecule fluorescent probes have been reported for labeling Kv7 channels, but these probe showed a little change with only 3- to 8-fold in the restricted rotation state over free rotation state, making them suffer the interference from background fluorescence. Thus, there is a lack of high sensitive probe for detecting the Kv channels.

The novel fluorescent probe for labeling Kv7 channel provides a pharmacological tool due to advantages of simplicity, sensitivity and visualization. Fluorescence and UV-vis absorption spectroscopy results demonstrate that the probe FPKv-3 exhibits excellent optical properties. The fluorescence intensity of FPKv-3 in the restricted state increased 183-fold in viscous media such as glycerol compared to that in the freely dispersed state. Fluorescence polarization (FP) values of FPKv-3 with Kv7.2/7.3 channels changed in a concentration-dependent manner with the Kd of 0.66 μM. In contrast, FP values of FPKv-3 with the membranes from non-transfected HEK293 cells decreased gently. Electrophysiological and confocal imaging experiments demonstrate that FPKv-3 possesses a high selectivity to Kv7 channels over other channels including TRPA1, TRPV3 and TRPV1, providing an effective chemical tool for understanding the roles of Kv7 channels.

We designed a novel small molecule fluorescent probe (FPKv-3) through connecting coumarin fluorophore and Kv7 ligand with a "long flexible chain". The fluorescence of FPKv-3 enhanced 183 times in glycerol than PBS solution, which may overcome the interference from background fluorescence. FPKv-3 probe could selectively imaging Kv7 channels in living cells, and has potential to be applied in high throughput screening of Kv7 agonists.