title: The alpha 1 and alpha 6 subunit subtypes of the mammalian GABA(A) receptor confer distinct channel gating kinetics.
abstract: The GABA(A) receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The alpha family is the largest, with six different subtypes. The alpha1 and alpha6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the alpha1 or alpha6 subunit was expressed in mammalian cells along with beta3 and gamma2L subunits and the kinetic properties examined with outside-out patch recordings. The alpha1 beta3 gamma2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the alpha6 beta3 gamma2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for alpha6 beta3 gamma2L, compared with the alpha1 beta3 gamma2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the alpha1 and alpha6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3-TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the alpha1 and alpha6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABA(A) receptors with different subunit composition.
authors.affiliation: Department of Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC 29208, USA. jfisher@gw.med.sc.edu
authors.forename: Janet L
authors.initials: JL
authors.lastname: Fisher
mesh_terms: D000818:Animals; D002460:Cell Line; D058785:GABA-A Receptor Agonists; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D012326:RNA Splicing; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013329:Structure-Activity Relationship; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics / Q000494:pharmacology
chemical_list: D058785:GABA-A Receptor Agonists; C546240:GABRA1 protein, human; C546171:GABRA6 protein, human; D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid