title: Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors.
abstract: The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs occurred as a single population of bursts (P(O) approximately 0.4-0.5) of moderate duration (approximately 33 ms) that could be described by schemes containing two shut and two open states for both GABA(A)Rs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.
authors.affiliation: CV Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology, Weill Medical College of Cornell University, NY, NY 10021, USA. ank2013@cornell.med.edu
authors.forename: Angelo
authors.initials: A
authors.lastname: Keramidas
authors.forename: Neil L
authors.initials: NL
authors.lastname: Harrison
mesh_terms: D000818:Animals; D002460:Cell Line; D003198:Computer Simulation; D004594:Electrophysiology; D018755:GABA Agonists / Q000494:pharmacology*; D058785:GABA-A Receptor Agonists; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008956:Models, Chemical; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000502:physiology; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D018755:GABA Agonists; D058785:GABA-A Receptor Agonists; C546240:GABRA1 protein, human; C546260:GABRA4 protein, human; C546279:GABRG2 protein, human; C546241:Gabra1 protein, mouse; C546242:Gabra1 protein, rat; C546261:Gabra4 protein, mouse; C546262:Gabra4 protein, rat; C502185:Gabrb2 protein, rat; C546280:Gabrg2 protein, mouse; C546281:Gabrg2 protein, rat; D007555:Isoxazoles; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid; C015542:gaboxadol