Abstract
The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.
Highlights
The GABAA receptor (GABAAR)2 of the ligand-gated ion channel (LGIC) superfamily (1, 2) is blocked by noncompetitive antagonists (NCAs) of widely diverse structures (3) including some of the most important insecticides (4) and, at another site, by Zn2ϩ (5)
To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extracellular half of the 3 homopentamer (11 to 20)
Induced disulfides formed with T16C, but there were few or none with M11C, T12C, and N15C. These findings show conformational flexibility/mobility in the M2 extracellular half 17 to 20 region interpreted as a deformed -like conformation in the open channel
Summary
Mutagenesis, Transfection, and Expression—Alignment of M2, M3, and flanking sequences of various LGICs are shown in. It was possible that the engineered M2 Cys might interact with the single endogenous Cys (Cys289) in M3 To avoid this possible complication, some dual mutations (13ЈC, 14ЈC, and 16ЈC to 20ЈC) were generated with Cys replaced by Ser at position 289 to give Cys-less M3. Similar protocols were followed here for single mutations to Cys, Ser, or Ala of M2 11Ј to 20Ј. The reaction was terminated by adding 10 mM N-ethylmaleimide and 1 mM EDTA Following this treatment sequence, sample buffer (3) was added with or without 10 mM dithiothreitol (DTT). DTT Reduction, Cu:phen Oxidation, and NCA Binding— Membranes as described above were subjected to reduction and oxidation to determine the effect of the disulfide on receptor NCA binding activity. Each sample was subjected to the [3H]EBOB binding assay. The M2 residues from 11Ј to 20Ј were mutated to Cys and the distances measured between sulfur centers
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