Hydrophobic pairwise interactions stabilize alpha-conotoxin MI in the muscle acetylcholine receptor binding site.

The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.

Molecular dissection of subunit interfaces in the nicotinic acetylcholine receptor.

Ligand binding sites in the muscle nicotinic acetylcholine receptor are generated by pairs of alpha and non-alpha subunits. The non-alpha subunits, gamma, delta and epsilon, contribute significantly to overall affinity of agonists and antagonists, and confer selectivity of these ligands for the two binding sites. By constructing chimeras composed of segments of the various non-alpha subunits and determining ligand selectivity, we have identified four loops, well separated in the linear sequence, that contribute to the non-alpha portion of the binding site. Studies of point mutations in these loops and labeling of engineered cysteines show that the peptide backbones of each non-alpha subunit fold into similar basic scaffolds. Studies of mutations of the peptide antagonists alpha-conotoxin M1 and ImI reveal pairs of residues in the binding site and the toxin that stabilize the complex.

Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the M2 domain of the acetylcholine receptor alpha subunit.

We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation alphaV249F in the propositus and mosaicism for alphaV249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, alphaV249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the alphaV249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.

Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation.

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.

Identification of residues in the adult nicotinic acetylcholine receptor that confer selectivity for curariform antagonists.

We identify residues in the epsilon and delta subunits of the adult nicotinic acetylcholine receptor that give the alphaepsilon and alphadelta binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing epsilon-delta subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileepsilon58/Hisdelta60 and Aspepsilon59/Aladelta61, 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 Ilegamma116/Valdelta118, Tyrgamma117/Thrdelta119, and Sergamma161/Lysdelta163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.

New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome.

Mutations in genes encoding the epsilon, delta, beta and alpha subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: epsilon L269F and delta Q267E in Patient 1, beta V266M in Patient 2, and alpha N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. epsilon L269F, delta Q267E and beta V266M occur in the second and alpha N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but delta Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. epsilon L269F, beta V266M, and alpha N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; epsilon L269F and alpha N217K enhance desensitization, and epsilon L269F and beta V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky, delta Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.

Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon 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 alpha-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.

Immunomagnetic separation of subpopulations of apolipoprotein A-I.

OBJECTIVE: This study was undertaken to measure the subfractions of high-density lipoprotein (HDL) in patients with diabetes or coronary artery disease and in normal control subjects. DESIGN: A new immunomagnetic separation technique was used to characterize the lipid profile in four groups: (1) control subjects, (2) patients with diabetes but no coronary artery disease (CAD), (3) those with CAD only, and (4) those with both diabetes and CAD. MATERIAL AND METHODS: To study the individual roles of the two discrete HDL subpopulations of particles--LpAI/AII (apolipoprotein [apo] A-I associated with A-II) and LpAI (apo A-I without A-II)--in lipoprotein metabolism, we developed an immunomagnetic separation technique using magnetic beads coated with antibodies to human apo A-II. The beads bind particles that contain both apo A-II and apo A-I and are precipitated by a magnetic field. LpAI levels were measured in the supernatant by performing an apo A-I radioimmunoassay. LpAI/AII levels were determined by subtracting the LpAI levels from total plasma apo A-I. RESULTS: In comparison with control subjects, patients with diabetes, CAD, or both had significantly decreased levels of LpAI/AII. LpAI levels were normal in patients with diabetes without CAD but significantly lower than control values in those with diabetes and CAD. CONCLUSION: Our findings suggest that both subpopulations of HDL particles have implications in the development of atherosclerosis in patients with and without diabetes.

A particle concentration fluorescence immunoassay for Lp(a).

The quantitation of Lp(a) by immunoassay presents a major technical problem, because the molecular mass of the (a) protein of Lp(a) can vary between 419,000 and 838,000 Da (Gaubatz et al. (1990) J. Lipid Res. 31, 603-612), and this variability is determined by at least 24 alleles of the (a) gene. In an attempt to overcome this problem, we have developed an assay that is independent of variation of the size of (a). The assay utilizes a mixture of monoclonal antibodies to (a) which do not react to plasminogen or to apolipoprotein (apo) B. These antibodies are bound to inert microscopic beads to capture the Lp(a) particles. Subsequently, a fluorescein-labeled monoclonal antibody to apo B is used for detection and quantitation. The assay is done with special microtiter plates containing filters so that the particles can be thoroughly washed after capture on the microbeads. Because Lp(a) particles contain only one apo B particle and the molecular weight of apo B is constant, the assay is not affected by variation in the size of apo(a). By binding the mixture of monoclonal antibodies to inert beads, it is possible to greatly increase the amount of antibody bound to an exposed surface and thus increase the sensitivity of the assay. A mixture of monoclonal antibodies can be used to increase the affinity of the capture step of the assay. The assay can be completed in 4 h and has a wide working range.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and quantification of apolipoproteins in addition to apo[a] and apo B-100 in human lipoprotein[a].

The protein moiety of Lp[a] is widely believed to consist of one molecule of apo B-100 and one molecule of apo[a] per particle, linked by at least one disulfide bond. In this study we have re-examined the composition of Lp[a] to determine if other less abundant apolipoproteins might be present. Analysis of Lp[a] by sodium dodecyl sulfate-polyacrylamide electrophoresis under reducing conditions showed bands corresponding to < 200 kD but > 50 kD, 40 kD, 26 kD, 23 kD and 9 kD when stained with silver. Western immunoblot analysis of three preparations of Lp[a] revealed the presence of apoE and apoD. Enzyme-linked immunoassays were used to quantify apoA-I, apoA-II, apoC-I, apoC-II, apoC-III, apoE and apo B-100 in Lp[a] and autologous LDL isolated from three healthy males. There is a significant amount of apoA-I in the Lp[a], although the levels varied widely among the different samples. ApoE concentrations were consistent in the three Lp[a] samples and were between 22 and 26% of relative apo B-100 concentrations. Relatively minor amounts of apoA-II and no apoCs were detectable in the three Lp[a] preparations. In contrast, the autologous LDL preparations contained relatively higher amounts of apoA-I, apoA-II, apoE, apoC-I, apoC-II and apoC-III. The identity of the multiple bands corresponding to < 200 kD and > 54 kD and 9 kD is not established.

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.

Separation of high-density lipoproteins into apolipoprotein E-poor and apolipoprotein E-rich subfractions by fast protein liquid chromatography using a heparin affinity column.

The aim of this paper is to describe a new methodology for the separation of human high-density lipoproteins (HDL) into apolipoprotein (apo) E-poor and apo E-rich subfractions by fast protein liquid chromatography (FPLC) using a heparin affinity column. Recoveries for apolipoproteins AI, AII, CI, CII, CIII, and E were 68.9, 74.7, 71.9, 73.5, 40.0, and 55.8%, respectively. We provide suggestive evidence that apo E-rich HDL is produced from apo E-poor HDL by the displacement of apo AI by apo E. Apo E-poor HDL was the predominant fraction. The molar ratio of apo E to apo AI in apo E-poor HDL was 0.02 and 0.01 for the subjects studied while in apo E-rich HDL it was 1.86 and 1.25. The molar ratios of the C apolipoproteins to apo AI are markedly different between the subfractions.

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.