Screening of ion channel receptor agonists using capillary electrophoresis-patch clamp detection with resensitized detector cells

Efficient techniques for identifying endogenous and synthetic ligands of io n channels are important in understanding neuronal communication and for sc reening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection wher e a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbac hol, and (-)-nicotine were fractionated and detected by patch-clamped pheoc hromcytoma detector cells. The high-conductance state of the nAChr during C E-PC detection was maintained aid repetitively resensitized using pulsed-fl ow superfusion with agonist;fi-ee buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the i ntrinsic high selectivity and sensitivity of membrane-expressed receptors a nd allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.