Differential protein mobility of the gamma-aminobutyric acid, type A, receptor alpha and beta subunit channel-lining segments.
The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.
Horenstein
Jeffrey
J
Departments of Physiology and Biophysics and of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA.
Riegelhaupt
Paul
P
Akabas
Myles H
MH
D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003975:Diazepam / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D005033:Ethylmaleimide / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D009068:Movement; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D004220:Disulfides; D021122:Protein Subunits; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D015032:Zinc; D003545:Cysteine; D005033:Ethylmaleimide; D003975:Diazepam; D004229:Dithiothreitol