State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABA_Aand Acetylcholine Receptor M3 Segments

Abstract

Construction of a GABA A receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABA A and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABA A M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues ␣1M266 (11Ј) and ␣1T267 (12Ј) were mutated to cysteine in either wild type or single M3 cysteine mutant (␣1V297C, ␣1A300C to ␣1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABAinduced currents in ␣1T267C-L301C and ␣1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in ␣1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the ␣-carbon separation was at most 5.6 Å. The reactive ␣1M3 residues (L301, F304, A305) lie on the same face of an ␣-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of ␣1M3 faces M2-␣1T267. In conjunction with the ACh structure, our data indicate that alignment of GABA A and ACh M3 requires a single gap in the GABA A M2-M3 loop. In the presence of GABA, oxidation of ␣1T267C-L301C and ␣1T267C-F304C had no effect, but oxidation of ␣1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-␣1T267 and M3-␣1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.

Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to γ-Aminobutyric Acid Type A Receptors

“…3B) that positions neither ␣1Met-236 nor ␤Met-286 at the interface between the subunits. The location of ␣1Met-236 at the ␤-␣ interface was consistent with other models based upon the nAChR structure (19,20), and as shown in Fig. 3, the recent identification (22) of the positions in ␣M1 that can be crosslinked to ␤M286C or ␤F289C is consistent with the homology model of Li et al (12) but not that of Hosie et al (16).…”

“…3, the recent identification (22) of the positions in ␣M1 that can be crosslinked to ␤M286C or ␤F289C is consistent with the homology model of Li et al (12) but not that of Hosie et al (16). Further support for this alignment comes from Jansen and Akabas (20), who used Cys mutagenesis and sulfhydryl cross-linking to orient the ␣M3 segment relative to ␣M2, which suggests proximity between ␤M2-Thr-262 and ␤M3 residues Leu-296, Tyr-299, and Ala-300, as in Fig. 3A.…”

“…For GABA A Rs and other members of the Cys-loop superfamily of neurotransmitter-gated ion channels, the transmembrane domain of each subunit is made up of a loose bundle of four ␣ helices (M1-M4), with M2 from each subunit contributing to the lumen of the ion channel and M4 positioned peripherally in greatest contact with lipid, as seen in the structures of the Torpedo nicotinic acetylcholine receptor (nAChR) (17) and in distantly related prokaryote homologs (18). However, uncertainties in the alignment of GABA A R subunit sequences relative to those of the nAChR result in alternative GABA A R homology models (12,19,20) that differ in the location of amino acids in the M3 and M4 membrane-spanning helices and in the M1 helix in some models (16,21 (16), is also consistent with cysteine substitution cross-linking studies (20,22), which define the proximity relations between amino acids in the ␣M1, ␣M2, ␣M3, and ␤M3 helices, and these results support the interpretation that the two residues photolabeled by [ 3 H]azietomidate are part of a single subunit interface binding pocket, whereas the steroid sensitivity determinants identified by mutagenesis neither are at the ␤/␣ subunit interface nor are contributors to a common binding pocket.…”

Spontaneous Mobility of GABAA Receptor M2 Extracellular Half Relative to Noncompetitive Antagonist Action

“…The species considered are human for GABA A R and rat for glycine receptor (GlyR) ␣ 1, serotonin type 3 A receptor (5-HT3 A R), and nAChR ␣ 1 . The subunit-specific number is given at the left for the first residue of each aligned sequence (7). Index numbers (see Footnote 3) for positioning in M2 are shown at the top.…”

“…The expression of ␤ 3 homomer was evaluated as the mean fluorescence value. Disulfide Cross-linking-Disulfide cross-linking profiles were determined for the single and dual mutants comparing spontaneous, oxidative, and reductive conditions (3,7,9). CuSO 4 was prepared as a 100 mM stock solution in water and o-phenanthroline (Sigma) as a 200 mM stock solution in ethanol.…”