title: Discrete M3-M4 intracellular loop subdomains control specific aspects of γ-aminobutyric acid type A receptor function.
abstract: The GABA type A receptor (GABA(A)R) is a member of the pentameric ligand gated ion channel (pLGIC) family that mediates ionotropic neurotransmission. Residues in the intracellular loop domain (ILD) have recently been shown to define part of the ion permeation pathway in several closely related members of the pentameric ligand gated ion channel family. In this study, we investigated the role the ILD of the GABA(A)R α1 subunit plays in channel function. Deletion of the α1 ILD resulted in a significant increase in GABA EC(50) and maximal current amplitude, suggesting that the ILD must be intact for proper receptor function. To test this hypothesis, we conducted a mutagenic screen of all amino acids harboring ionizable side chains within this domain to investigate the contribution of individual charged residues to ion permeation. Using macroscopic and single channel voltage-clamp recording techniques, we found that mutations within a subdomain of the α1 ILD near M3 altered GABA apparent affinity; interestingly, α1(K312E) exhibited reduced partial agonist efficacy. We introduced point mutations near M4, including α1(K383E) and α1(K384E), that enhanced receptor desensitization. Mutation of 5 charged residues within a 39-residue span contiguous with M4 reduced relative anion permeability of the channel and may represent a weak intracellular selectivity filter. Within this subdomain, the α1(K378E) mutation induced a significant reduction in single channel conductance, consistent with our hypothesis that the GABA(A)R α1 ILD contributes directly to the permeation pathway.
authors: Departments of Anesthesiology and Pharmacology, Emory University, Atlanta, Georgia 30322.
authors: Kate K
authors: KK
authors: O'Toole
authors: Departments of Anesthesiology and Pharmacology, Emory University, Atlanta, Georgia 30322. Electronic address: ajenki2@emory.edu.
authors: Andrew
authors: A
authors: Jenkins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017353:Gene Deletion; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014101:Torpedo
chemical_list: D008024:Ligands; D011963:Receptors, GABA-A