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 ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free 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 intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.
Farre
C
C
Department of Chemistry, Göteborg University, Sweden.
Sjöberg
A
A
Jardemark
K
K
Jacobson
I
I
Orwar
O
O
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D004353:Drug Evaluation, Preclinical; D019075:Electrophoresis, Capillary; D007473:Ion Channels / Q000819:agonists*; D008024:Ligands; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D051381:Rats
D007473:Ion Channels; D008024:Ligands; D000109:Acetylcholine