Interaction of Non-competitive Blockers within the γ-Aminobutyric Acid Type A Chloride Channel Using Chemically Reactive Probes as Chemical Sensors for Cysteine Mutants

Perret, Philippe; Sarda, Xavier; Wolff, Mark A.; Wu, Tai-Teh; Bushey, Dean F.; Goeldner, Maurice

doi:10.1074/jbc.274.36.25350

Cited by 62 publications

(65 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on these studies, it has been suggested that the picrotoxin binding site lies between the 2Ј and 6Ј positions of TMII (29,32). However, more recent work has shown that picrotoxin can also protect a cysteine engineered into the extracellular aspect of TMII (17Ј position) from irreversible modification by chemically reactive picrotoxin-related compounds (33). Although not definitive, these data are consistent with earlier suggestions that picrotoxin and related ligands may interact with multiple sites (7,10,34,35).…”

Section: Discussionsupporting

confidence: 79%

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

Dibas

Gonzales

Das

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The central nervous system convulsant picrotoxin (PTX) inhibits GABA A and glutamate-gated Cl ؊ channels in a use-facilitated fashion, whereas PTX inhibition of glycine and GABA C receptors displays little or no use-facilitated block. We have identified a residue in the extracellular aspect of the second transmembrane domain that converted picrotoxin inhibition of glycine ␣1 receptors from non-use-facilitated to usefacilitated. In wild type ␣1 receptors, PTX inhibited glycine-gated Cl ؊ current in a competitive manner and had equivalent effects on peak and steady-state currents, confirming a lack of use-facilitated block. Mutation of the second transmembrane domain 15-serine to glutamine (␣1(S15Q) receptors) converted the mechanism of PTX blockade from competitive to non-competitive. However, more notable was the fact that in ␣1(S15Q) receptors, PTX had insignificant effects on peak current amplitude and dramatically enhanced current decay kinetics. Similar results were found in ␣1(S15N) receptors. The reciprocal mutation in the ␤2 subunit of ␣1␤2 GABA A receptors (␣1␤2(N15S) receptors) decreased the magnitude of use-facilitated PTX inhibition. Our results implicate a specific amino acid at the extracellular aspect of the ion channel in determining use-facilitated characteristics of picrotoxin blockade. Moreover, the data are consistent with the suggestion that picrotoxin may interact with two domains in ligand-gated anion channels.Glycine receptors belong to a superfamily of ligand-gated chloride channels that include GABA A 1 receptors, GABA C receptors, and glutamate-gated chloride channels (1). In native tissue, glycine receptors exist as either ␣ homomers or ␣␤ heteromers (1). They comprise five subunits (usually three ␣ subunits and two ␤ subunits) arranged asymmetrically around the ion pore. Each subunit is made up of a large extracellular N-terminal region, four transmembrane domains (TM), and a large cytoplasmic domain; TMII forms the channel lumen (2). Glycine receptors are targets of therapeutics such as anesthetics as well as toxins like the central nervous system convulsant picrotoxin (1).Picrotoxin inhibits all known anionic ligand-gated Cl Ϫ channels (3-5). The mechanism of action and the exact location of picrotoxin binding are still unknown (6 -12). However, several studies have indicated that TMII is the probable site for picrotoxin action (6, 13-22) (Fig. 1). For example, the TMII of the glycine ␤ subunit was found to be responsible for conferring resistance to picrotoxin in heteromeric glycine ␣ n ␤ receptors (n ϭ 1-3) (6). Subsequent work has defined the existence of a phenylalanine residue at the 6Ј position of the TMII glycine ␤ subunit in conferring insensitivity to picrotoxin (16). In addition, other TMII residues (2Ј and 19Ј) have also been implicated directly or indirectly in the mechanism by which picrotoxin inhibits these channels (13, 15,16). The mutations at positions 2Ј and 19Ј have been shown to affect the type of the inhibition (competitive versus non-competitive) by picrotoxin ...

show abstract

Section: Discussionsupporting

confidence: 79%

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

Dibas

Gonzales

Das

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…␤ 3 homopentamer mutants A-1ЈC, R0ЈC, and R0ЈS block binding, perhaps because of proximity to A2Ј. Sulfhydryl modification at V1ЈC impedes (19). The involvement of 3Ј, directly or by influencing the neighboring A2Ј, is shown by L3ЈF at ␤ 3 of the ␣ 1 ␤ 3 receptor almost abolishing TBPS and PTX binding (20).…”

Section: Discussionmentioning

confidence: 99%

“…The molecular localization of the NCA site defined here (Fig. 1B) was first indicated by mutagenesis studies (16) as A2Ј (17)(18)(19)(20), T6Ј (21,22), and L9Ј (23,24) in the cytoplasmic half of the transmembrane 2 domain of the channel (Fig. 2).…”

mentioning

confidence: 99%

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Chen

Durkin

Casida

2006

Proc. Natl. Acad. Sci. U.S.A.

150

151

View full text Add to dashboard Cite

Several major insecticides, including ␣-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human ␤3 homopentameric GABAA receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the ␤3 homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1 C, A2 C, T6 C, and L9 C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands ␤3 homopentamer ͉ transmembrane 2 ͉ insecticide ͉ disulfide trapping ͉ receptor model P est insect control in the past 60 years was achieved, in part, by application of Ͼ3 billion (3 ϫ 10 9 ) pounds of polychlorocycloalkane insecticides, including cyclodienes (e.g., ␣-endosulfan and dieldrin), lindane and its isomers, and others, which are now highly restricted or banned except for endosulfan and some uses of lindane (1-3). One of the replacement compounds is the phenylpyrazole fipronil. All of these insecticides and the botanical picrotoxinin (PTX) have widely diverse chemical structures but appear to act at the same nerve target. It is therefore important to understand how these compounds work in mammals and insects, or how they do not work when resistant insect strains appear.The GABA-gated chloride channel is the target for the insecticides and toxicants referred to above based on radioligand binding and electrophysiology studies (3)(4)(5)(6)(7)(8)(9)(10) (Fig. 1A). All of these compounds act in mammals and insects as noncompetitive antagonists (NCAs) to block chloride flux so the target is referred to as the GABA receptor NCA-binding site. Vertebrate GABA receptors consist of ␣, ␤, ␥, , and other subunits in various combinations, for example, ␣ 1 ␤ 2 ␥ 2 as a heteropentamer and 1 as a homopentamer (13-15). The molecular localization of the NCA site defined here (Fig. 1B) was first indicated by mutagenesis studies (16) as A2Ј (17-20), T6Ј (21, 22), and L9Ј (23, 24) in the cytoplasmic half of the transmembrane 2 domain of the channel (Fig. 2). Drosophila resistant to dieldrin (RDL) have a mutation conferring GABA receptor insensitivity identified as A2ЈS (17). The NCA target of the GABA A receptor requires a ␤ subunit, and a ␤ 3 homopentamer is sufficient for binding (9,26). Importantly, the ␤ 3 subunit from human brain, when expressed in insect Sf9 cells, assembles to form a receptor sensitive to all of the important GABAergic insecticides (9) and, surprisingly, reproduces the insecticide sensitivity and structureactivit...

show abstract

“…This approach has proven to be broadly applicable to other ligand-gated ion channels to probe successfully agonist-(21), antagonist- (22,23), allosteric modulator- (24), and channel blocker-binding sites (25). It consists in a proximity-accelerated chemical reaction between a thiol-reactive ATP analogue and a single free cysteine mutant engineered in the putative ATPbinding site (Fig.…”

mentioning

confidence: 99%

Agonist trapped in ATP-binding sites of the P2X2 receptor

Jiang

Lemoine

Martz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATPderived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATPbinding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.affinity labeling | chemical modification | purinergic receptor P 2X receptors are oligomeric ATP-gated ion channels selective to cations (1) and are involved in physiological processes as diverse as synaptic transmission, the response to inflammation, and pain perception (2). Upon ATP binding, structural rearrangements of the subunit interface (3-5) lead to the opening of the ion channel (6-8), but the entire molecular sequence of events that couple ATP binding to channel opening remains unknown. The recent X-ray structure of the P2X4 receptor in a closed resting state represents in this regard a decisive step (9). It confirms the trimeric stoichiometry of the ion channel, in agreement with the fact that there are three activatable ATP-binding sites (10), and provides a structural context to interpret functional data (11).Early studies based on mutagenesis data have identified highly conserved extracellular residues important for ATP function and have proposed that ATP binding occurs through the extracellular domain (12-19), presumably at the subunit interface (15,17). When mapped on the crystal structure, most of these residues are observed to line a large and deep intersubunit cavity shaped like an open "jaw" and located approximately 45 Å away from the ion channel domain (9). This observation thus suggests that these residues participate in ATP binding; however, the crystal structure was solved in the absence of ATP, and therefore no direct evidence support this hypothesis to date.We used the proximity-dependent "tethering" approach (20) to localiz...

show abstract

Interaction of Non-competitive Blockers within the γ-Aminobutyric Acid Type A Chloride Channel Using Chemically Reactive Probes as Chemical Sensors for Cysteine Mutants

Cited by 62 publications

References 31 publications

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

Identification of a Novel Residue within the Second Transmembrane Domain That Confers Use-facilitated Block by Picrotoxin in Glycine α1 Receptors

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Agonist trapped in ATP-binding sites of the P2X2 receptor

Contact Info

Product

Resources

About