title: A rapid agonist application system for fast activation of ligand-gated ion channels.
abstract: The synaptic delay between neurotransmitter release across the synaptic cleft and activation of neurotransmitter gated ion channels is less than a ms. Nicotinic acetylcholine receptors (nAChRs), like many other classes of ligand-gated ion channels, are comprised of different protein subunits forming a variety of receptors with different activation and desensitization kinetics and pharmacological sensitivities. To measure and fully characterize ligand-gated ion channel currents accurately, one must apply agonists in a fraction of a ms and repeatedly at various concentrations without any prior desensitization of the receptors. In this paper, we describe an economical, easy to assemble and operate rapid drug application system. The drug applicator system consists of a parallel array of three pinch valves, which allow either agonist or wash solution into a theta tube. Solution exchanges of 0.16 ms can be achieved. In transfected cells, ACh elicited α4β2 nicotinic currents with mean rise times of 55±13 ms. We recorded α7 nAChRs, which desensitize very rapidly, and obtained very fast rise times of 19±2 ms. With this novel drug applicator, agonists can be applied repeatedly without any loss of current. Hence, complete dose-response relations can be obtained for even α7 nAChRs, which are very sensitive to desensitization caused by agonist exposure on a ms time scale. The drug application system can also be extended to the study of ligand-gated ion channels in brain slices. The theta tube valve-driven drug applicator system can be applied to study other ligand-gated ion channels including glutamate and GABA receptors.
authors.affiliation: Department of Biology, University of Victoria, Victoria, British Columbia, Canada. pragya@uvic.ca
authors.forename: Pragya
authors.initials: P
authors.lastname: Komal
authors.forename: Gareth
authors.initials: G
authors.lastname: Evans
authors.forename: Raad
authors.initials: R
authors.lastname: Nashmi
mesh_terms: D000818:Animals; D001921:Brain / Q000502:physiology; D002478:Cells, Cultured; D004594:Electrophysiology / Q000295:instrumentation* / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D058446:Ligand-Gated Ion Channels / Q000819:agonists* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000187:drug effects*; D011978:Receptors, Nicotinic / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects*
chemical_list: D058446:Ligand-Gated Ion Channels; D011978:Receptors, Nicotinic