Pharmacological and Physiological Characterization of Murine Homomeric β3 GABA A Receptors

The polypeptide snake toxin ␣-bungarotoxin (BTX) has been used in hundreds of studies on the structure, function, and development of the neuromuscular junction because it binds tightly and specifically to the nicotinic acetylcholine receptors (nAChRs) at this synapse. We show here that BTX also binds to and blocks a subset of GABA A receptors (GABAARs) that contain the GABAAR ␤3 subunit. These results introduce a previously unrecognized tool for analysis of GABA ARs but may complicate interpretation of some studies on neuronal nAChRs.acetylcholine ͉ neurotransmitter ͉ synapse P erhaps the best understood of all synapses is the vertebrate skeletal neuromuscular junction (1). Although the accessibility, simplicity, and large size of this synapse are often viewed as its principle advantages, another important factor has been the availability of a highly versatile ligand for the nicotinic acetylcholine receptors (nAChRs) that are the critical component of its postsynaptic membrane. This ligand, ␣-bungarotoxin (BTX), is a 74-aa polypeptide derived from the venom of the banded krait, Bungarus multicinctus. BTX binds quasiirreversibly and highly specifically to the nAChRs at the neuromuscular junction and in electric organs of Torpedoes and rays (2, 3). An additional advantage is that it is readily derivatized without loss of activity; it has been conjugated to agarose for affinity-purification of AChRs (4), to 125 I for quantification of AChRs (5), and to fluorophores for tracking of AChRs during synaptogenesis (ref. 6; see refs. 3 and 7 for reviews of early and recent work, respectively).BTX binds not only to the ␣1 subunit contained in muscle nAChRs but also to a subset of neuronal nAChRs; it binds those containing the ␣7-␣10 subunits but not those containing the ␣2-␣6 subunits (8, 9). As at the neuromuscular junction, studies of the structure, function, and development of neuronal cholinergic synapses have benefited from BTX.In studies on both muscle and neuronal cholinergic synapses, a guiding assumption has been that BTX binds only to nAChRs. During the course of studies to label neurotransmitter receptors that had been tagged with a BTX-binding motif (10), we obtained evidence that challenged this premise. We show here that BTX binds to a subset of GABA A receptors (GABA A Rs). In particular, BTX blocks GABA A Rs that contain interfaces between adjacent ␤3 subunits. Results BTX Binds to a GABAAR Subunit in Heterologous Cells.In initial studies, we found that fluorophore-conjugated BTX stained human embryonic kidney (HEK 293) cells that had been transfected with a cDNA encoding the rat GABA A R ␤3 subunit (Fig. 1a). This staining was not seen in untransfected cells (Fig. 1a). Staining by fluorophore-conjugated BTX was blocked by preincubation with unconjugated BTX, whereas staining was seen when cells were incubated successively with BTX, an affinitypurified antibody to BTX, and fluorophore-conjugated second antibody (Fig. 1a and data not shown). Similar results were obtained in several cell types, and specific ...

Section: Analysis Of Homooligomeric Gaba ␤3 Receptors In Xenopus Oocycontrasting

confidence: 99%

Section: Btx Blocks Chloride Conductance Through Gabaar ␤3 Receptorssupporting

confidence: 92%

The cholinergic antagonist α-bungarotoxin also binds and blocks a subset of GABA receptors

McCann

Bracamontes

Steinbach

et al. 2006

“…The tight protein packing in the 2Ј position (31) may account for the weak dimer formation by limiting the required flexibility and close proximity for disulfide bond formation. Disulfides are not formed with 0Ј, 1Ј, 4Ј, 5Ј, 7Ј, 8Ј, and 10Ј, indicating they are probably not in the pore or have low (28), suggesting that the ␤ 3 homopentamer of the present investigation assumes the spontaneous open state (32). Homology of the GABA receptor ␤ 3 homopentamer with the nicotinic acetylcholine receptor (33) indicates the narrowest gating region of the pore is between 9Ј and 14Ј, suggesting the positioning of L9ЈC in the pore (24,31).…”

Section: Discussionmentioning

confidence: 68%

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

Chen

Durkin

Casida

2006

149

151

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...

“…To identify the domain of the ␥2 subunit that mediates the lysosomal targeting of GABA A Rs we exploited the ability of GABA A R ␤3 subunits to assemble into pentameric cell surface homomeric ion channels, a property that depends on residues within the N terminus of this subunit (28,29). As measured by antibody labeling, internalized ␤3 subunits showed a similar distribution to ␣␤ receptors with respect to GFP-Rab7 (33.8 Ϯ 3.7%) ( Fig.…”

Section: Identification Of a Sorting Motif In The Gabaar ␥2 Subunit Thatmentioning

confidence: 99%

Ubiquitin-dependent lysosomal targeting of GABA _A receptors regulates neuronal inhibition

Arancibia-Cárcamo

Yuen

Muir

et al. 2009

Self Cite

The strength of synaptic inhibition depends partly on the number of GABA A receptors (GABAARs) found at synaptic sites. The trafficking of GABA ARs within the endocytic pathway is a key determinant of surface GABA AR number and is altered in neuropathologies, such as cerebral ischemia. However, the molecular mechanisms and signaling pathways that regulate this trafficking are poorly understood. Here, we report the subunit specific lysosomal targeting of synaptic GABA ARs. We demonstrate that the targeting of synaptic GABAARs into the degradation pathway is facilitated by ubiquitination of a motif within the intracellular domain of the ␥2 subunit. Blockade of lysosomal activity or disruption of the trafficking of ubiquitinated cargo to lysosomes specifically increases the efficacy of synaptic inhibition without altering excitatory currents. Moreover, mutation of the ubiquitination site within the ␥2 subunit retards the lysosomal targeting of GABA ARs and is sufficient to block the loss of synaptic GABA ARs after anoxic insult. Together, our results establish a previously unknown mechanism for influencing inhibitory transmission under normal and pathological conditions. endocytosis ͉ trafficking ͉ ischemia ͉ ion channels ͉ synapse T he strength of inhibitory synapses is a major determinant of neuronal processing and excitability and can be determined by the number of ␥-amino butyric acid type A receptors (GABA A Rs) found at synaptic sites (1-3). Moreover, in a number of neuropathologies, including status epilepticus and in vitro and in vivo models of cerebral ischemia, the rapid downmodulation of synaptic GABA A Rs has been reported to contribute to pathological disinhibition, excitotoxicity, and cell death (4-8). However, the molecular mechanisms and signaling pathways that regulate GABA A R trafficking, under normal or pathological conditions, remain unclear.GABA A Rs undergo significant rates of endocytosis, a process that can rapidly modify receptor number at synapses (9). Upon internalization, receptors are either reinserted into the plasma membrane or targeted for degradation (10). The balance between these two processes is critical for determining the number of receptors expressed at synapses and hence neuronal excitability (11). However, the regulatory mechanisms that control this endocytic sorting of synaptic GABA A Rs remain unknown.The trafficking of membrane proteins can be regulated via the covalent attachment of the 76 amino acid (aa) peptide, ubiquitin, or ubiquitin-like proteins to lysine residues within target proteins (12,13). A number of studies on the role of ubiquitination in regulating synaptic function under normal conditions and in pathological conditions such as ischemia have focused on the ubiquitin-proteasome system (14-18). However, within the endocytic pathway, mono-ubiquitination (or the addition of short ubiquitin chains) is a key signal that results in the specific targeting of cargo to late endosomes for subsequent lysosomal degradation (12,19,20). The significance of ubiquitinati...