Nina Bren

Congenital Myasthenic Syndrome Caused by Decreased Agonist Binding Affinity Due to a Mutation in the Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Molecular dissection of subunit interfaces in the acetylcholine receptor: Identification of determinants of α-Conotoxin M1 selectivity

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.

Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors

Structure-function studies have identified key functional motifs in the acetylcholine receptor, including residues that contribute to the ion channel and to the ligand-binding sites. Little is known, however, about determinants of channel gating kinetics. To identify structural correlates of gating, we examined the structural basis of the fetal-to-adult decrease in channel open time conferred by the presence of the epsilon subunit in place of the gamma subunit. By constructing chimeras composed of segments of the epsilon and gamma subunits, we show that the main determinant of this kinetic change is a 30 residue segment of a predicted amphipathic helix located between transmembrane domains M3 and M4. Further subdividing the amphipathic helix revealed that either multiple residues or its overall conformation confers this regulation of channel kinetics. We also show that L440 and M442, conserved residues within M4 of the gamma subunit, contribute to long duration openings characteristic of the fetal receptor.

Ligand-binding domain of an alpha 7-nicotinic receptor chimera and its complex with agonist.

The α7 acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α7 AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α7. We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α7 AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α7 AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure–function relationships of α7 AChRs.

Lysine scanning mutagenesis delineates structural model of the nicotinic receptor ligand binding domain.

Nicotinic acetylcholine receptors (AChR) and their relatives mediate rapid chemical transmission throughout the nervous system, yet their atomic structures remain elusive. Here we use lysine scanning mutagenesis to determine the orientation of residue side chains toward core hydrophobic or surface hydrophilic environments and use this information to build a structural model of the ligand binding region of the AChR from adult human muscle. The resulting side-chain orientations allow assignment of residue equivalence between AChR subunits and an acetylcholine binding protein solved by x-ray crystallography, providing the foundation for homology modeling. The resulting structural model of the AChR provides a picture of the ACh binding site and predicts novel pairs of residues that stabilize subunit interfaces. The overall results suggest that lysine scanning can provide the basis for structural modeling of other members of the AChR superfamily as well as of other proteins with repeating structures delimiting a hydrophobic core.

Naturally Occurring Mutations at the Acetylcholine Receptor Binding Site Independently Alter ACh Binding and Channel Gating

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, ɛN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αɛ site. The second CMS mutation, ɛD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. ɛD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, ɛN182Y localizes to the interface with the α subunit, and ɛD175 to the entrance of the ACh binding cavity. Both ɛN182Y and ɛD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring ɛN182 and ɛD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera.

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³⁶ and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹⁸⁴ from loop-C of α7, while Asp³⁰ of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹⁸⁴. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹⁸⁴ with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

Identification of Residues in the Adult Nicotinic Acetylcholine Receptor That Confer Selectivity for Curariform Antagonists

We identify residues in the ε and δ subunits of the adult nicotinic acetylcholine receptor that give the αε and αδ binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing ε-δ subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileε58/Hisδ60 and Aspε59/Alaδ61, responsible for DMT site selectivity in the adult receptor. The two determinants contribute approximately equally to the binding site and interact in contributing to the site. Exchange of these residues from one subunit to the other exchanges the affinities of the resulting binding sites. These determinants in the adult receptor are far from those that confer site selectivity in the fetal receptor; determinants in the fetal receptor are Ileγ116/Valδ118, Tyrγ117/Thrδ119, and Serγ161/Lysδ163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.

A rapid apolipoprotein E radioimmunoassay using solid-phase staphylococcus protein. Use of pooled plasma as a secondary standard

A rapid apolipoprotein E (apo E) radioimmunoassay, which requires a total of 24 hour incubation as compared to the usual 3-5 days, has been developed in our laboratory. Solid phase staphylococcus protein A was used to separate bound and unbound labeled antigen. Use of a pooled plasma (quality control sample) as a secondary standard to reduce interassay variation was also described.

Quantification of Human Plasma Apolipoproteins C-I, C-II, and C-III by Radioimmunoassays

We have developed radioimmunoassays for the quantification of apolipoproteins (apo) C-I, C-II, and C-III in human plasma. The apo C proteins were isolated from very low-density lipoproteins of patients with hypertriglyceridemia, fractionated on a Sephacryl column, and purified by diethylaminoethyl cellulose anion-exchange chromatography followed by reverse-phase fast protein liquid chromatography. The assays were sensitive, specific, and reproducible, and the standards demonstrated parallel immunoreactivity with plasma samples. Patients with hypertriglyceridemia (triglyceride level more than 2,200 mg/liter)--14 patients with diabetes and 12 with type V hyperlipoproteinemia--were compared with age- and sex-matched control subjects. In comparison with the control groups, levels of apoproteins C-I, C-II, and C-III were significantly increased in both disease groups, but the ratios of the C peptides to triglycerides were significantly lower, an indication of a relative deficiency of C apoproteins in hypertriglyceridemic states. Independent radioimmunoassays for each of the C apolipoproteins would help to study their individual roles in triglyceride-rich lipoprotein metabolism.