Human α4β2 Acetylcholine Receptors Formed from Linked Subunits

Zhou, Yan; Nelson, Mark E.; Kuryatov, Alexander; Choi, Catherine H.; Cooper, Jay M.; Lindstrom, Jon

doi:10.1523/jneurosci.23-27-09004.2003

Cited by 160 publications

(211 citation statements)

References 35 publications

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“…A potential hazard of subunit concatenation is the possibility that not all subunits in the concatemer will participate in the formation of the complex, leading to erroneous results. For example, either ␣ 4 -␤ 2 or ␤ 2 -␣ 4 nicotinic acetylcholine receptor subunit dimers were unexpectedly found to form functional receptors (34). It was shown that dimers could be incorporated into two separate pentamers, leading to a receptor dimer held together by the linker.…”

Section: Discussionmentioning

confidence: 99%

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Stewart

Haerteis

Diakov

et al. 2011

Journal of Biological Chemistry

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The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the ␣-, ␤-and ␥-subunits. We show that for ␣-, ␤-and ␥-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that ␣␤␥-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.The epithelial sodium channel (ENaC) 5 is a member of the ENaC/degenerin superfamily, which also includes acid-sensing ion channels (ASICs; reviewed in Ref. 1). ENaC mediates Na ϩ entry across the apical membranes of many absorptive epithelia, including the alveolar epithelium, salivary duct, distal colon, and sweat glands. ENaC plays a particularly important role in Na ϩ transport across the aldosterone-sensitive distal nephron (reviewed in Ref. 2), which is responsible for the regulation of Na ϩ and K ϩ excretion, and thus plays an important role in the control of blood pressure. ENaC is the target of potassiumsparing diuretics such as amiloride and spironolactone used in the treatment of cardiovascular disease. Whereas amiloride is a direct blocker of the channel, the mineralocorticoid receptor antagonist spironolactone inhibits ENaC activity by preventing its stimulation by aldosterone.ENaC is a heteromultimer containing three homologous subunits (␣, ␤, and ␥), which have 30 -40% amino acid identity (3, 4). A fourth ENaC subunit, ␦-ENaC, has been cloned from a human kidney library (5). In heterologous expression systems, ␦-ENaC has functional similarities with ␣-ENaC, but its physiological role is still unclear (6). Each ENaC subunit contains two transmembrane domains, a large extracellular loop, and short intracellular N and C termini. Full ENaC activity requires coexpression of all three subunits (4). In the past, it has been proposed that ENaC is assembled from either four (7-9) or eight to nine subunits (10, 11). However, the recently published crystal structure of ASIC 1a (12) suggests that ENaC is likely to be a trimer (13), although this has not been demonstrated.We have developed a method, based on AFM imaging, for determining the stoichiometry and arrangement of subunits within multimeric proteins (reviewed in Ref. 14). The method involves engineering specific epitope tags onto each protein subunit and expressing the proteins exogenously in a suitable cell line (usually tsA 201). Membrane fractions from the transfected cells are solubilized in detergent, and the proteins are isola...

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Section: Discussionmentioning

confidence: 99%

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Stewart

Haerteis

Diakov

et al. 2011

Journal of Biological Chemistry

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“…3E). These residues likely contribute to αβ and ββ interfaces in α4β2 nAChRs (21,28,29). In the human β2 subunit, these residues on the complementary face of the binding site correspond to V111, F119, and L121 (loop E) and W57 (loop D).…”

Section: Structure-guided Mutagenesis Analysis Of Varenicline Interacmentioning

confidence: 99%

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

Billen

Spurný

Brams

et al. 2012

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

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Partial agonists of the α4β2 nicotinic acetylcholine receptor (nAChR), such as varenicline, are therapeutically used in smoking cessation treatment. These drugs derive their therapeutic effect from fundamental molecular actions, which are to desensitize α4β2 nAChRs and induce channel opening with higher affinity, but lower efficacy than a full agonist at equal receptor occupancy. Here, we report X-ray crystal structures of a unique acetylcholine binding protein (AChBP) from the annelid Capitella teleta, CtAChBP, in complex with varenicline or lobeline, which are both partial agonists. These structures highlight the architecture for molecular recognition of these ligands, indicating the contact residues that potentially mediate their molecular actions in α4β2 nAChRs. We then used structure-guided mutagenesis and electrophysiological recordings to pinpoint crucial interactions of varenicline with residues on the complementary face of the binding site in α4β2 nAChRs. We observe that residues in loops D and E are molecular determinants of desensitization and channel opening with limited efficacy by the partial agonist varenicline. Together, this study analyzes molecular recognition of smoking cessation drugs by nAChRs in a structural context. addiction | cys-loop receptor | ligand-gated ion channel

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“…Given that α5 gene deletion did not change cytisine-sensitive [ 3 H]-epibatidine binding (Figure 3), we suspect that both α4β2 and α4α5β2 nAChRs are normally expressed in striatal dopaminergic nerve terminals and that α4β2 nAChRs replace the α4α5β2 nAChRs in the α5 null mutant mice. It has been repeatedly demonstrated that α4β2 nAChRs expressed in Xenopus oocytes or cultured cells can assemble in two stoichiometric forms with differing sensitivities to activation by agonists [108,109,110]. In neurons there is evidence for two forms of α4β2 receptors with functional differences [111, and MJ Marks, unpublished data); however, these two forms may have similar binding affinities (MJ Marks, unpublished data).…”

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confidence: 99%

The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum

Grady

Salminen

Laverty

et al. 2007

Biochemical Pharmacology

232

223

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This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [ 125 I]-α-bungarotoxin and [ 3 H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and α-conotoxin MII (α-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the α6 subunit (α4α6β2β3, α6β2β3, α6β2) and bind α-CtxMII with high affinity. One of these three subtypes (α4α6β2β3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for α-CtxMII (α4β2, α4α5β2) are somewhat more numerous than the α6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions. Alterations in nicotinic cholinergic receptor (nAChRs) number or function have been implicated in psychopathologies such as anxiety, attention deficit hyperactivity disorder, depression, schizophrenia (reviewed in [1,2,3]), at least one form of familial epilepsy [4], and Parkinson's and Alzheimer's diseases [5]. It is not particularly surprising that nAChRs might play important roles in modulating several human diseases given that binding sites for nicotinic ligands such as [ 125 I]-α-bungarotoxin [6,7,8], [ 3 H]-nicotine [7,8] Corresponding author: Sharon R Grady e-mail: sharon.grady@colorado.edu phone: 303-492-9677 fax: 303-492-8063 mailing address: Institute for Behavioral Genetics University of Colorado 447UCB Boulder, CO 80309. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered w...

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Human α4β2 Acetylcholine Receptors Formed from Linked Subunits

Cited by 160 publications

References 35 publications

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Atomic Force Microscopy Reveals the Architecture of the Epithelial Sodium Channel (ENaC)

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum

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