title: Two beta-rich structural domains in GABA(A) receptor alpha(1) subunit with different physical properties: Evidence for multidomain nature of the receptor.
abstract: The type A gamma-aminobutyric acid (GABA(A)) receptor is a major inhibitory neurotransmitter-gated ion channel. Previously, we identified a membrane-proximal beta-rich (MPBR) domain in fragment C166-L296 of GABA(A) receptor alpha(1) subunit, forming nativelike pentamers. In the present study, another structural domain, the amino-terminal domain, was shown to exist in the fragment Q28-E165. The secondary structures of both fragments were beta-rich as measured using FTIR spectroscopy and estimated from the CD spectra to be 42% and 51% beta-strand for Q28-E165 and C166-L296, respectively. The CD spectrum of the combined fragment Q28-L296 was additive of the spectra of the two fragments. In addition, denaturation curves of both fragments were characteristic of cooperative transitions, supporting their domainlike nature. C166-L296 required 6.5 M of guanidine chloride for total denaturation, therefore it is extraordinarily stable, more so than Q28-E165. Moreover, effects of detergent on the molecular masses of Q28-E165 and C166-L296, as monitored using laser-scattering spectroscopy, indicated that intermolecular interactions were much more significant in C166-L296 than in Q28-E165. Effects of pH on their molecular masses suggested that ionic forces were involved in these interactions. Together the results show that the two adjacent fragments form independent folding units, MPBR and amino-terminal domains, different in secondary structure content, denaturation profile, and polymerization status, and suggest that the former may play a more important role in receptor assembly and that the extraordinary stability may underlie its intrinsic tendency to form oligomers. More significantly, the present study has provided direct evidence for the long-postulated multidomain nature of this family of receptors.
authors.affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Shi
authors.forename: Shui Ying
authors.initials: SY
authors.lastname: Tsang
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: James N
authors.initials: JN
authors.lastname: Sturgis
authors.forename: Hong
authors.initials: H
authors.lastname: Xue
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007834:Lasers; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry; D011963:Receptors, GABA-A / Q000737:chemistry*; D012542:Scattering, Radiation; D017550:Spectroscopy, Fourier Transform Infrared; D014508:Urea / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D021122:Protein Subunits; D011963:Receptors, GABA-A; D014508:Urea; D019791:Guanidine