HTS Assay for Ca 3 T-type Channels Using FLIPR (RMI)

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DESCRIPTION (provided by applicant): T-type Ca2+ channels, encoded by three genes (Cav3.1, 3.2 and 3.3, or alpha1-G, H, and I), are heterogeneously expressed in the brain and many peripheral organs such as the heart and vascular smooth muscle. They play a role as the pacemaker that regulates spontaneous neuronal activity, cardiac rhythm and vascular tone. T-channels have been a drug target for absence epilepsy and hypertension. The conventional anti-absence epilepsy drug ethosuximide produces weak inhibition of the T-channel. The anti-hypertensive drug, mibefradil, potently blocks the T channel but was withdrawn from the market due to drug interaction. Inhibition of T-channels by 619C89 and SB-209712 may contribute to the neuroprotective effects of the compounds; however, these drugs are non-specific and produce many side effects. Phenotypic studies on the Cav3.1 or 3.2 gene knockout mice have shed light on the biological role of T-channels and suggest the T-channel could be a potential target for therapeutic intervention in pathological pain and cardiovascular diseases. There is no doubt that subtype-selective and potent Cav3 modulators should produce more specific pharmacological actions with fewer side effects. High throughput screening (HTS) of a large compound library is the initial step to identify such a novel compound. Standard electrophysiological techniques are not suitable for primary HTS. A recent study using single cell Ca2+ imaging techniques has demonstrated that manipulation of extracellular Ca2+ significantly changes the intracellular Ca2+ ([Ca2+]j) in the HEK293 cells expressing human Cav3.2 channels (Cav3.2-HEK293). Detection of [Ca2+]j indirectly measures Ca2+ entry through spontaneous opening of the Cav3.2 channels (i.e., window currents). This method is amenable to HTS. In a pilot study we tested the feasibility using the Ca2+ sensitive dye fluo-4/AM and measured with a fluorometric imaging plate reader (FLIPRR) in a 96-well format. In this proposal, our specific aims are to (1) develop a 384-well HTS format through optimization of assay conditions and automation of procedures such as the integration of a robotic system and automated data analysis, and (2) validate this fluorometric method by comparison of the potency of a large number of ion channel inhibitors determined using both the FLIPR assay and whole-cell voltage-clamp recording techniques. The proposed fluorometric method will provide a high throughput and quantitative assay for the primary screening of compounds against three subtypes of Cav3 T-type channels.