Abstract
GABA(A) receptors are ligand-gated ion channels formed by the pseudosymmetrical assembly of five homologous subunits around the central channel axis. The five M2 membrane-spanning segments largely line the channel. In the present work we probed the water surface accessibility of the beta(1) subunit M2 segment using the substituted cysteine accessibility method. We assayed the reaction of the negatively charged sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS(-)), by its effect on subsequent currents elicited by EC(50) and saturating GABA concentrations. pCMBS(-), applied with GABA, reacted with 14 of the 19 residues tested. At the M2 cytoplasmic end from 2' to 6' only beta(1)A252C (2') and beta(1)T256C (6') were pCMBS(-)-reactive in the presence of GABA. We infer that the M2 segments are tightly packed in this region. Toward the extracellular half of M2 all residues from beta(1)T262C (12') through beta(1)E270C (20') reacted with pCMBS(-) applied with GABA. We infer that this region is highly mobile and loosely packed against the rest of the protein. Based on differences in pCMBS(-) reaction rates two domains can be distinguished on the putative channel-lining side of M2. A faster reacting domain includes the 2', 9', 12', 13', and 16' residues. The slower reacting face contains the 6', 10', and 14' residues. We hypothesize that these may represent the channel-lining faces in the closed and open states and that gating involves an 80-100 degrees rotation of the M2 segments. These results are consistent with the loose packing of the M2 segments inferred from the structure of the homologous Torpedo nicotinic acetylcholine receptor.
Highlights
The GABAA1 receptors are the major inhibitory neurotransmitter receptors in the central nervous system [1]
To probe the protein packing around the M2 segment we assayed the accessibility of cysteines (Cys) substituted for the GABAA receptor 1 subunit M2 segment residues to react with the sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate. pCMBSϪ is a rigid, negatively charged molecule that will fit into a right cylinder that is ϳ6 Å in diameter and ϳ10 Å in length. pCMBSϪ reacts with Cys and covalently couples HgC6H4SO3Ϫ onto the sulfhydryl. pCMBSϪ is membrane-impermeant [20, 21] and reacts 1,000 times faster with ionized thiolates (–SϪ) than with thiols (–SH) [22]; a reaction is much more likely with water-accessible Cys that can ionize
We infer that the Cys at these positions were covalently modified by pCMBSϪ
Summary
Mutagenesis and Oocyte Expression—PCR mutagenesis of the rat 1 subunit in the pGEMHE vector, in vitro mRNA transcription, and preparation and injection of Xenopus oocytes were performed as described previously [27]. PCMBSϪ was applied for 1 min at 0.5 mM either in the absence or in the presence of a saturating concentration of GABA This combination of time and concentration was chosen because they were the maximal concentration and duration that caused no significant increase in the leak conductance of uninjected Xenopus oocytes. Given the variability of responses, application of a reagent must cause a net change in current greater than ϳ30% to be significantly different from wild type by a one-way analysis of variance (ANOVA) (for n between 3 and 6) With this threshold and the pCMBSϪ reaction conditions (0.5 mM applied for 1 min), if complete reaction caused 100% inhibition of the GABA-induced current we would detect reactive positions with a second order reaction rate Ͼ12 liters/mol/s. Reaction rates are given as the mean Ϯ S.E
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