Design And Evaluation Of A Fluorescent Probe Targeting HERG Channel

hERG channel is a target of drug cardiotoxicity evaluation and is closely associated with cell-cycle of some tumors,including gastric cancer,colon cancer,leukemia and glioblastoma.Several methods such as patch clamp,radio-ligand,and fluorescence-based assay,were used to evaluate hERG channels in different biological systems.However,each of them has disadvantages such as low throughput,radioactive contamination or low selectivity.Therefore,a simple assay with high throughput,outstanding specificity and excellent sensitivity is urgent needed for understanding the roles of hERG channels in biological systems.Hence,we described a simple fluorescent probe CBH for imaging of hERG channels by employing a novel targeting group,4-benzylaniline.The specificity of CBH was confirmed with electrophysiology,confocal imaging and immunofluorescence.Results showed that both solvatochromism and molecular rotation regulated the fluorescent emission of CBH and make the fluorescent intensity change remarkably,thus improving the sensitivity to hERG channels.In addition,it was proved the CBH probe has potential for assessing the expression and function of hERG channels in cells and tissues,especially in tumor cells bearing hERG protein at a low level.Objective: The purpose of this study was to label hERG channel expressed on cell membrane and quantitatively measure the expression of hERG channel via a fluorescent probe CBH based on a hERG targeting group 4-benzylaniline,which is expected to be a useful chemical tool for studies of the biological roles of hERG channels in cardiovascular system and tumor cell-cycle.Methods: The fluorescence mechanism of CBH was researched by fluorescence spectroscopy and ultraviolet spectroscopy,and the function of CBH on hERG channels was identified through electrophysiology.Then a fluorescence polarization was utilized to evaluate the binding affinity of CBH for hERG channel.The ability of labeling hERG channel expressed on cell membrane and quantitatively measuring the expression of hERG channel were testified via confocal.Results: The study of fluorescence spectroscopy and ultraviolet spectroscopy showed that the fluorescent emissions of CBH are regulated by the synergy between solvatochromism and rotation of rotors.In electrophysiology section,it was proved that4-benzylaniline has an activation effect while coumarin has an inhibition effect on hERG channels,which indicated 4-benzylaniline played a crucial role in activation of hERG channels.Then we further utilized the co-incubation of CBH with ZBH to verify the electrophysiological results that 4-benzylaniline was a targeting group to hERG channel,and measured the overlap values between immunofluorescence experiment of hERG with anti-Kv11.1 antibody and cell image of CBH to evaluate the staining of hERG protein expressed on cell membrane.At last,we further explore the application potential of CBH for quantitative analysis the low expression of hERG protein,which was also identified by Western Blot.Conclusions: We designed a “turn on” hERG probe CBH based on a novel targeting group 4-benzylaniline and the synergeticregulation mechanism between solvatochromism and molecular rotation.The probe CBH showed high selectivity and excellent sensitivity to hERG protein,and was successfully applied in imaging of hERG channels in He La cells with low expression.Thus,the probe CBH is expected to be a useful chemical tool for studies of the biological roles of hERG channels in cardiovascular system and tumor cell-cycle,and its novel targeting group can provide a basis for further design of other fluorescent probes,inhibitor or activator to hERG channels.