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Myles H Akabas

Publications and source records attributed to Myles H Akabas.

5 recordsLinked to original sources
PubMed2004

Loose protein packing around the extracellular half of the GABA(A) receptor beta1 subunit M2 channel-lining segment.

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.

4-Chloromercuribenzenesulfonate↗
PubMed2004

Defining the propofol binding site location on the GABAA receptor.

The GABAA receptor is a target of many general anesthetics. The low affinity of general anesthetics has complicated the search for the location of anesthetic binding sites. Attention has focused on two pairs of residues near the extracellular ends of the M2 and M3 membrane-spanning segments, alpha1Ser270/beta2Asn265 (15' M2) and alpha1Ala291/beta2Met286 (M3). In the 4-A resolution acetylcholine receptor structure, the aligned positions are separated by approximately 10 A. To determine whether these residues are part of a binding site for propofol, an intravenous anesthetic, we probed propofol's ability to protect cysteines substituted for these residues from modification by the sulfhydryl-specific reagent p-chloromercuribenzenesulfonate (pCMBS-). pCMBS- reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABAAreceptor, we needed to establish a reference state of the receptor to compare reaction rates in the absence and presence of propofol. We compared reaction rates in the presence of GABA with those in the presence of propofol +GABA. The GABA concentration was reduced to give a similar fraction of the maximal GABA current in both conditions. Propofol protected, in a concentration-dependent manner, the cysteine substituted for beta2Met286 from reaction with pCMBS-. Propofol did not protect the cysteine substituted for the aligned alpha1 subunit position or the 15' M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the betasubunit M3 segment.

4-Chloromercuribenzenesulfonate↗
PubMed2002

GABA(A) receptor M2-M3 loop secondary structure and changes in accessibility during channel gating.

The gamma-aminobutyric acid type A (GABA(A)) receptor M2-M3 loop structure and its role in gating were investigated using the substituted cysteine accessibility method. Residues from alpha(1)Arg-273 to alpha(1)Ile-289 were mutated to cysteine, one at a time. MTSET(+) or MTSES(-) reacted with all mutants from alpha(1)R273C to alpha(1)Y281C, except alpha(1)P277C, in the absence and presence of GABA. The MTSET(+) closed-state reaction rate was >1000 liters/mol-s at alpha(1)N274C, alpha(1)S275C, alpha(1)K278C, and alpha(1)Y281C and was <300 liters/mol-s at alpha(1)R273C, alpha(1)L276C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C. These two groups of residues lie on opposite sides of an alpha-helix. The fast reacting group lies on a continuation of the M2 segment channel-lining helix face. This suggests that the M2 segment alpha-helix extends about two helical turns beyond alpha(1)N274 (20'), aligned with the extracellular ring of charge. At alpha(1)S275C, alpha(1)V279C, alpha(1)A280C, and alpha(1)A284C the reaction rate was faster in the presence of GABA. The reagents had no functional effect on the mutants from alpha(1)A282C to alpha(1)I289C, except alpha(1)A284C. Access may be sterically hindered possibly by close interaction with the extracellular domain. We suggest that the M2 segment alpha-helix extends beyond the predicted extracellular end of the M2 segment and that gating induces a conformational change in and/or around the N-terminal half of the M2-M3 loop. Implications for coupling ligand-evoked conformational changes in the extracellular domain to channel gating in the membrane-spanning domain are discussed.

Amino Acid Sequence↗
PubMed2002

Structural evidence that propofol stabilizes different GABA(A) receptor states at potentiating and activating concentrations.

The GABA(A) receptor is a target of many general anesthetics, such as propofol. General anesthetic binding sites are distinct from the GABA binding sites. At low concentrations, the anesthetics potentiate the currents induced by submaximal GABA concentrations. At higher concentrations the anesthetics directly activate GABA(A) receptors. In contrast, benzodiazepines, such as diazepam, only potentiate currents induced by submaximal GABA concentrations. Channel kinetic studies suggest that these drugs stabilize different receptor states. We previously showed that the accessibility of the anionic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS(-)) applied extracellularly to cysteines substituted for residues in the GABA(A) alpha1 subunit M3 membrane-spanning segment was state-dependent. The subset of pCMBS(-)-accessible, M3 segment cysteine mutants acts as a reporter for receptor conformation. Here we show that pCMBS(-), applied in the presence of a potentiating concentration of propofol, reacts with a subset of alpha1 subunit, M3 segment, cysteine-substitution mutants (Y294C, V297C, I302C, F304C). In the presence of a directly activating concentration of propofol pCMBS(-) reacts with a different subset of the M3 cysteine-substitution mutants (Y294C, S299C, I302C, E303C, A305C). These subsets are distinct from the subsets of M3 cysteine-substitution mutants that are reactive with pCMBS(-) in the absence and presence of GABA and in the presence of diazepam. We hypothesize that distinct subsets of reactive residues represent distinct conformations or ensembles of conformations of the receptor. These results provide structural evidence for at least five distinct receptor states, three nonconducting states, resting, diazepam-bound and potentiating propofol-bound, and two conducting-desensitized states, the activating propofol-bound and GABA-bound states.

4-Chloromercuribenzenesulfonate↗