γ-Aminobutyric Acid Increases the Water Accessibility of M3 Membrane-Spanning Segment Residues in γ-Aminobutyric Acid Type A Receptors

Abstractγ-Aminobutyric acid type A (GABAA) receptors are members of the ligand-gated ion channel gene superfamily. Using the substituted cysteine accessibility method, we investigated whether residues in the α1M3 membrane-spanning segment are water-accessible. Cysteine was substituted, one at a time, for each M3 residue from α1Ala291 to α1Val307. The ability of these mutants to react with the water-soluble, sulfhydryl-specific reagent pCMBS− was assayed electrophysiologically. Cysteines substituted for α1Ala291 and α1Tyr294 reacted with pCMBS− applied both in the presence and in the absence of GABA. Cysteines substituted for α1Phe298, α1Ala300, α1Leu301, and α1Glu303 only reacted with pCMBS− applied in the presence of GABA. We infer that the pCMBS− reactive residues are on the water-accessible surface of the protein and that GABA induces a conformational change that increases the water accessibility of the four M3 residues, possibly by inducing the formation of water-filled crevices that extend into the interior of the protein. Others have shown that mutations of α1Ala291, a water-accessible residue, alter volatile anesthetic and ethanol potentiation of GABA-induced currents. Water-filled crevices penetrating into the interior of the membrane-spanning domain may allow anesthetics and alcohol to reach their binding sites and thus may have implications for the mechanisms of action of these agents

Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants

We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257–261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 1 subunit. The residues alpha 1Val257 and alpha 1Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of alpha 1V257C and alpha 1T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as alpha 1Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to alpha 1T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Co-application of picrotoxin prevented the sulfhydryl reagents from reacting with alpha 1V257C but did not prevent reaction with the more extracellular residue alpha 1T261C. Picrotoxin protection of alpha 1V257C may be due to steric block by picrotoxin bound in the channel at the level of alpha 1Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which alpha 1V257C was accessible to sulfhydryl reagent

The Location of a Closed Channel Gate in the GABAA Receptor Channel

Considerable controversy surrounds the location of the closed channel gate in members of the Cys-loop receptor family of neurotransmitter-gated ion channels that includes the GABAA, glycine, acetylcholine, and 5-HT3 receptors. Cysteine-accessibility studies concluded that the gate is near the cytoplasmic end of the channel in acetylcholine and GABAA receptors but in the middle of the 5-HT3A receptor channel. Zn2+ accessibility studies in a chimeric 5-HT3-ACh receptor suggested the gate is near the channel's cytoplasmic end. In the 4-Å resolution structure of the acetylcholine receptor closed state determined by cryoelectron microscopy, the narrowest region, inferred to be the gate, is in the channel's midsection from 9' to 14' but the M1–M2 loop residues at the channel's cytoplasmic end were not resolved in that structure. We used blocker trapping experiments with picrotoxin, a GABAA receptor open channel blocker, to determine whether a gate exists at a position more extracellular than the picrotoxin binding site, which is in the vicinity of α1Val257 (2') near the channel's cytoplasmic end. We show that picrotoxin can be trapped in the channel after removal of GABA. By using the state-dependent accessibility of engineered cysteines as reporters for the channel's structural state we infer that after GABA washout, with picrotoxin trapped in the channel, the channel appears to be in the closed state. We infer that a gate exists between the picrotoxin binding site and the channel's extracellular end, consistent with a closed channel gate in the middle of the channel. Given the homology with acetylcholine and 5-HT3 receptors there is probably a similar gate in those channels as well. This does not preclude the existence of an additional gate at a more cytoplasmic location

Modular Design of Cys-loop Ligand-gated Ion Channels: Functional 5-HT3 and GABA ρ1 Receptors Lacking the Large Cytoplasmic M3M4 Loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the α-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT3A) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT3A-glvM3M4 receptors were comparable to 5-HT3A wild type. The channels remained cation-selective but the 5-HT3A-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT3 and acetylcholine receptors, significantly attenuated 5-HT–induced currents with wild-type 5-HT3A but not 5-HT3A-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-ρ1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains

Defining the propofol binding site location on the GABAA receptor

ABSTRACT The GABA A 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, ␣ 1 Ser270/␤ 2 Asn265 (15Ј M2) and ␣ 1 Ala291/␤ 2 Met286 (M3). In the 4-Å resolution acetylcholine receptor structure, the aligned positions are separated by ϳ10 Å. 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 Ϫ ). pCMBS Ϫ reacted with cysteines substituted at the four positions in the absence and presence of GABA. Because propofol binding induces conformational change in the GABA A receptor, 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 ϩ 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 ␤ 2 Met286 from reaction with pCMBS Ϫ . Propofol did not protect the cysteine substituted for the aligned ␣ 1 subunit position or the 15Ј M2 segment Cys mutants in either subunit. We infer that propofol may bind near the extracellular end of the ␤ subunit M3 segment

Conformational variability of the glycine receptor M2 domain in response to activation by different agonists

Models describing the structural changes mediating cys-loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric α1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19’ or 22’ positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19’C increased by ~20% and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and β-alanine were weak partial agonists at the a1R19’C GlyR, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue-shift in the spectral emission peak. The inhibitors, strychnine and picrotoxin, elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or β-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22’C residue. Thus, results from two separate labelled residues support the conclusion that the GlyR M2 domain responds with distinct conformational changes to activation by different agonists

5-HT3 receptor ion size selectivity is a property of the transmembrane channel, not the cytoplasmic vestibule portals

5-HT3A receptors select among permeant ions based on size and charge. The membrane-associated (MA) helix lines the portals into the channel’s cytoplasmic vestibule in the 4-Å resolution structure of the homologous acetylcholine receptor. 5-HT3A MA helix residues are important determinants of single-channel conductance. It is unknown whether the portals into the cytoplasmic vestibule also determine the size selectivity of permeant ions. We sought to determine whether the portals form the size selectivity filter. Recently, we showed that channels functioned when the entire 5-HT3A M3–M4 loop was replaced by the heptapeptide M3–M4 loop sequence from GLIC, a bacterial Cys-loop neurotransmitter gated ion channel homologue from Gloebacter violaceus. We used homomeric 5-HT3A receptors with either a wild-type (WT) M3–M4 loop or the chimeric heptapeptide (5-HT3A–glvM3M4) loop, i.e., with or without portals. In Na+-containing buffer, the WT receptor current–voltage relationship was inwardly rectifying. In contrast, the 5-HT3A–glvM3M4 construct had a negative slope conductance region at voltages less than −80 mV. Glutamine substitution for the heptapeptide M3–M4 loop arginine eliminated the negative slope conductance region. We measured the relative permeabilities and conductances of a series of inorganic and organic cations ranging from 0.9 to 4.5 Å in radius (Li+, Na+, ammonium, methylammonium, ethanolammonium, 2-methylethanolammonium, dimethylammonium, diethanolammonium, tetramethylammonium, choline, tris [hydroxymethyl] aminomethane, and N-methyl-d-glucamine). Both constructs had measurable conductances with Li+, ammonium, and methylammonium (size range of 0.9–1.8-Å radius). Many of the organic cations >2.4 Å acted as competitive antagonists complicating measurement of conductance ratios. Analysis of the permeability ratios by excluded volume theory indicates that the minimal pore radius for 5-HT3A and 5-HT3–glvM3M4 receptors was similar, ∼5 Å. We infer that the 5-HT3A size selectivity filter is located in the transmembrane channel and not in the portals into the cytoplasmic vestibule. Thus, the determinants of size selectivity and conductance are located in physically distinct regions of the channel protein

Cyclic Nucleotide–Gated Channels: Pore Topology Studied through the Accessibility of Reporter Cysteines

In voltage- and cyclic nucleotide–gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the α subunit of cyclic nucleotide–gated channels from bovine rod, the pore loop is formed by the residues R345–S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, ∼50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible