title: Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.
abstract: Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.
authors.affiliation: Department of Chemical and Biological Engineering, Physical Chemistry, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Granfeldt
authors.forename: Jon
authors.initials: J
authors.lastname: Sinclair
authors.forename: Maria
authors.initials: M
authors.lastname: Millingen
authors.forename: Cecilia
authors.initials: C
authors.lastname: Farre
authors.forename: Per
authors.initials: P
authors.lastname: Lincoln
authors.forename: Owe
authors.initials: O
authors.lastname: Orwar
mesh_terms: D000818:Animals; D002460:Cell Line; D004594:Electrophysiology; D007700:Kinetics; D008024:Ligands; D018408:Patch-Clamp Techniques / Q000379:methods*; D011485:Protein Binding; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D015091:beta-Alanine / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D008024:Ligands; D011963:Receptors, GABA-A; D015091:beta-Alanine; D005680:gamma-Aminobutyric Acid