ABSTRACT
Transient receptor potential A1 (TRPAl) is a cold sensitive cation channel, which could also be activated by various pungent compounds. As a transduction channel in a number of sensory modalities, TRPAl expressing in heterogonous systems serves to provide great convenience in pharmacological analysis and functional investigation. Due to cellular toxicity, establishment of stable TRPAl cell line has always been challenging. Nevertheless, the first stable human embryonic kidney (HEK-293) cell line with un-controlled expression of TRPAl was successfully established. It was also confirmed that this stable cell line retained TRPAl expression for more than 25 passages in culture. The functional analysis of the cell response verified the stability and specificity of this novel recombinant TRPAl cell line. Altogether, the data indicated this TRPAl-HEK cell line would be a useful tool for functional analysis of TRPAl and for the development of high throughput screening (HTS) compatible assay in the effort to identify TRPAl modulators.
ABSTRACT
We established a cell based high throughput screening model by calcium assay on fluorometric imaging plate reader for finding modulators of TRPV3. The TRPV3 expression vector was transfected into HEK-293 and stable cell line expressing TRPV3 was selected with antibiotics. Upon TRPV3 specific modulators stimulated, pharmacological characteristics of TRPV3 over expression cell line were detected by calcium assay on fluorometric imaging plate reader. Assay conditions were optimized and stability of the model was observed. The reliability and accuracy of application to 96 and 384 well format high throughput screening were also evaluated. A stable HEK-293 cell line highly expressing TRPV3 was established. TRPV3 specific modulators could modulate calcium signal through TRPV3 in dose dependent manner. Optimized screening condition was established by assay development. This model is stable and sensitive, and meets the requirement of high throughput screening by Z'factor validation and Spiking test. This cell model can be applied to screening TRPV3 modulators by calcium assay.