Figure 6.

Investigation of auxiliary methods to increase choline-induced membrane potential to facilitate the HTS screen. (A) The cholinergic enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine (ACh) from choline. ChAT was transiently transfected into HEK293T cells and produced the cytoplasmic protein. (B) ChAT transfected in HEK293T cells was capable of producing ACh as measured by HPLC analysis. (C) ChAT transfection into HEK293 cells reduced the basal choline level of the cells (**n = 4, p < 0.01). (D) The choline utilizing enzymes, CHKA and ChAT, significantly increased ³H-choline uptake in cotransfected HEK293T cells (***n = 6, p < 0.001). (E) The cotransfection of LV-AA CHT (white bar) with ChAT (X-hatch bar) did not increase the response to the choline-induced membrane potential assay. (F) Inhibition of the organic cation transport (OCT) system with clonidine⁶⁵ reduced the ³H-choline uptake increase seen following ChAT cotransfection with CHT LV-AA. Significant change was determined by Student’s t-test analyses of all trials n = 3–5 for all assays and reported under values as *–*** for p values less than 0.05–0.001.