Development of a Membrane Potential Assay to detect Sodium Channel Toxins

الملخص

The FLIPR membrane potential (FMP) dye ( Molecular Devices) is a high throughput optical screening tool for cell based assay. A membrane potential assay was designed to detect sodium channel toxins. This study aimed to develop a membrane potential assay to detect Na+ channel toxins, by using FLIPR membrane potential dye to monitor changes in Na+ flux. Neuroblastoma SH-SY-5Y cell line and fluorescence plate reader (Tecan) was involved in this study. the assay was set, by finding the best FMP dye form (red or blue), the best 96 wells microplate  in terms of its color ( clear or black microplate) and the optimum cell density that  give an accurate fluorescence signal. Testing validation of  designed assay by sodium channel activators; KCL and veratridine, in addition to sodium channel blockers; tetradotoxin and procainamide hydrochloride, investigating effect of Indian red scorpion venom on sodium channel, to find out whether it is activation or inhibition. The assay detected procainamide hydrochloride effects on membrane potential. However, it appeared that the assay was less sensitive to tetradotoxin. That could be due to concentrations of provided TTX stock solution. Effects of  Indian red scorpion venom    (Mesobuthus tamulus) were successfully investigated by the assay. The venom has activator effects, as it showed an increase in fluorescence that was related to persistent activation of sodium channel and delay of its inactivation. Treatment of SH-SY-5Y neuroblastoma cell line with Indian red scorpion venom followed by veratridine, caused much more fluorescence increase than treatment of the cells with the venom only .The assay successfully detected sodium channels activator toxins such as veratridine and sodium channels blockers toxins such as  procainamide hydrochloride. Activity of Indian red scorpion venom (Mesobuthus tamulus) investigated showed that the venom contains sodium channel activators.